Mucin 15 is expressed in human placenta and suppresses invasion of trophoblast-like cells in vitro

BACKGROUND: Trophoblast invasion is crucial for the development of normal placentas. Mucins are suggested to be involved in cancer invasion. However, the function of mucins in trophoblast invasion has never been reported. This study was to investigate the expression of mucin (MUC) 15 in human placenta and its role in trophoblast invasion. METHODS: MUC15 mRNA in human tissues was analyzed by Northern blot. MUC15 mRNA and protein in human placenta were detected by real-time RT-PCR and Western blot, respectively. The distribution of MUC15 was revealed by immunohistochemistry. The effects of MUC15 on trophoblast invasion in vitro were analyzed by matrigel invasion assay in human choriocarcinoma JAR and JEG-3 cells. RESULTS: MUC15 was expressed most highly in human placenta. MUC15 mRNA and protein increased with gestational age (P < 0.05, first versus third trimester). Immunohistochemistry showed that MUC15 protein was expressed by both cytotrophoblasts and syncytiotrophoblasts, especially at the apical membrane of syncytiotrophoblasts. In addition, MUC15 was found to be present in the glandular epithelium of the decidua. Overexpression of MUC15 substantially decreased matrigel invasion of JAR and JEG-3 cells by 87.5 +/- 1.1 and 83.8 +/- 5.7%, respectively, versus control, which was closely associated with an increase in mRNA expression of tissue inhibitors of metalloproteinases (TIMP)-1 and TIMP-2. Knockdown of MUC15 with small interfering RNA significantly reversed these effects (P < 0.05). CONCLUSIONS: Differential expression of MUC15 in human placentas may play a critical role in the regulation of trophoblast invasion.     

Effects of Notch2 and Notch3 on Cell Proliferation and Apoptosis of Trophoblast Cell Lines

Aims: To investigate the effect of Notch2 and Notch3 on cell proliferation and apoptosis of two trophoblast cell lines, BeWo and JAR.Methods: Notch2 and Notch3 expression in BeWo and JAR cells was upregulated or downregulated using lentivirus-mediated overexpression or RNA interference. The effect of Notch2 and Notch3 on cell proliferation was assessed by the CCK-8 assay. The effect of Notch2 and Notch3 on the apoptosis of BeWo and JAR cells was evaluated by flow cytometry using the Annexin V-PE Apoptosis kit. Lentivirus-based overexpression vectors were constructed by cloning the full-length coding sequences of human Notch2 and Notch3 C-terminally tagged with GFP or GFP alone (control) into a lentivirus-based expression vector. Lentivirus-based gene silencing vectors were prepared by cloning small interfering sequences targeting human Notch2 and Notch3 and scrambled control RNA sequence into a lentivirus-based gene knockdown vector. The effect of Notch2 and Notch3 on cell proliferation was assessed by the CCK-8 assay. And the effect of Notch2 and Notch3 on the apoptosis of BeWo and JAR cells was evaluated by flow cytometry using the Annexin V PE Apoptosis kit.Results: We found that the downregulation of Notch2 and Notch3 gene expression in BeWo and JAR cells resulted in an increase in cell proliferation, while upregulation of Notch3 and Notch2 expression led to a decrease in cell proliferation. Moreover, the overexpression of Notch3 and Notch2 in BeWo and JAR cells reduced apoptosis in these trophoblast cell lines, whereas apoptosis was increased in the cells in which the expression of Notch3 and Notch2 was downregulated.Conclusions: Notch2 and Notch3 inhibited both cell proliferation and cell apoptosis in BeWo and JAR trophoblast cell lines.     

Calreticulin associates with non-HLA-A,-B class I proteins in the human choriocarcinoma cell lines JEG-3 and BeWo.

Human placental trophoblast expresses as unusual repertoire of major histocompatibility complex (MHC) class I products that appears to reflect the unique role of this epithelium in mediating feto-maternal relations during pregnancy. Trophoblast is devoid of human leucocyte antigen (HLA)-A,-B antigens but can express one or more non-HLA-A,-B class I proteins. The human choriocarcinoma cell lines JEG-3, BeWo and JAR are widely used as models to study trophoblast. During attempts to isolate non-HLA-A,-B class I from JEG-3 and BeWo by immunoaffinity chromatography using a monoclonal antibody to beta 2-microglobulin we observed a 55,000 MW protein co-purifying with class I. N-terminal amino acid sequencing and immunoblotting using a specific antiserum identified this product as calreticulin, a molecule recently shown to be involved in the assembly of classical class I in human B-lymphoblastoid cells. In our hands JEG-3 and BeWo were found to express 45,000 MW non-HLA-A,-B class I proteins while the 40,000 MW HLA-G product was identified only in JEG-3. Our data suggest that calreticulin associates with non-HLA-A,-B class I heterodimers and with free 45,000 MW non-HLA-A,-B class I H chains in JEG-3. JAR was found to be devoid of detectable class I H chains but contained beta 2-microglobulin and calreticulin. However, calreticulin-beta 2-microglobulin complexes were not detected in JAR. Calreticulin and class I were apparently co-localized within the endoplasmic reticulum of JEG-3 cells whereas only class I was expressed at the cell surface. These studies demonstrate that calreticulin is associated with non-HLA-A,-B class I products in human choriocarcinoma cells.     

The expression of human leukocyte antigen-G on trophoblasts abolishes the growth-suppressing effect of interleukin-2 towards them

PROBLEM: We have shown the attenuated human leukocyte antigen (HLA)-G expression on trophoblasts and an aberrant expression of interleukin (IL)-2, a cytotoxic cytokine, in decidual tissue in preeclampsia, where deteriorated trophoblastic invasion into decidual layers may constitute a crucial pathogenesis. We hypothesized that the absence of HLA-G might make trophoblasts susceptible to compromise by IL-2. METHOD OF STUDY: We analyzed the growth of HLA-G-negative and positive cell lines, all of which possessed IL-2 receptors, in the culture with or without IL-2 supplementation. RESULTS: The proliferation of HLA-G positive trophoblastic cell lines (BeWo and JEG-3) was not influenced by the addition of IL-2, whereas a HLA-G-negative trophoblastic cell line (JAR) exhibited significantly decreased proliferation when cultured with IL-2. Interestingly, the transfection of JAR cells with HLA-G completely eliminates the growth-inhibitory effect of IL-2. CONCLUSION: The expression of HLA-G may commit trophoblasts to evade cell damage by IL-2, which may be relevant to maternal tolerance of the fetus during pregnancy and its derangement as exemplified by preeclampsia.     

Cyclosporin A enhances the ability of trophoblasts to displace the activated human umbilical vein endothelial cell monolayers

Transformation of the spiral arteries including the displacement of vascular endothelial cells by extravillous trophoblasts is an essential prerequisite to normal placentation. However, the activated endothelial cells resist the invasion of trophoblasts, which contributes to the pathologies of some pregnant disorders. Our previous studies have demonstrated that Cyclosporin A (CsA) promotes the migration and invasion of human first-trimester trophoblasts. In the present study, we further investigated whether CsA could promote the ability of trophoblasts to displace the activated human umbilical vein endothelial cell (HUVEC) monolayers and the possible molecular mechanisms. Human choriocarcinoma Jar cells were used as a model of invasive trophoblasts. CsA pretreated JAR cells (red) were added to HUVEC monolayers (green) activated with either necrotic JAR cells or tumor necrosis factor alpha (TNFα). The ability of JAR cells to displace HUVECs from the monolayers was examined by confocal microscopy. The effects of CsA on Titin and E-cadherin expression, matrix metalloproteinases (MMPs) activity and CXCL12 secretion of JAR cells were evaluated by western blot, gelatin zymography and enzyme-linked immunosorbent assay (ELISA), respectively. We found that CsA pretreatment increased the ability of JAR cells to displace activated HUVECs from the monolayers. However, the displacement was reduced by untreated JAR cells. Moreover, CsA pretreatment up-regulated Titin expression, down-regulated E-cadherin expression, improved MMP2 and MMP9 activity, and increased the CXCL12 secretion in JAR cells. These results indicate that CsA may improve the trophoblast invasion to activated HUVEC monolayers through different downstream targets, and ultimately, improve the transformation and remodeling of spiral arteries.     

Human leucocyte antigen (HLA) expression of primary trophoblast cells and placental cell lines,determined using single antigen beads to characterize allotype specificities of anti-HLA antibodies

Human trophoblast cells express an unusual repertoire of human leucocyte antigen (HLA) molecules which has been difficult to define. Close homology between and extreme polymorphism at the classical HLA class-I (HLA-I) loci has made it difficult to generate locus-specific monoclonal antibodies (mAbs). The problem of defining an antibody''s reactivity against the thousands of existing HLA-I allotypes has often made it impossible to determine the HLA bound by a mAb in biological samples from a normal outbred population. Here we have used commercially available beads coated with individual HLA-I to characterize experimentally the reactivity of nine mAb against 96 common HLA-I allotypes. In conjunction with donor HLA-I genotyping, we could then define the specific HLA molecules bound by these antibodies in normal individuals. We used this approach to analyse the HLA expression of primary trophoblast cells from normal pregnancies; the choriocarcinoma cells JEG-3 and JAR; and the placental cell lines HTR-8/SVneo, Swan-71 and TEV-1. We confirm that primary villous trophoblast cells are HLA null whereas extravillous trophoblast cells express HLA-C, HLA-G and HLA-E, but not HLA-A, HLA-B or HLA-DR molecules in normal pregnancy. Tumour-derived JEG-3 and JAR cells reflect extravillous and villous trophoblast HLA phenotypes, respectively, but the HLA repertoire of the in vitro derived placental cell lines is not representative of either in vivo trophoblast phenotype. This study raises questions regarding the validity of using the placental cell lines that are currently available as model systems for immunological interactions between fetal trophoblast and maternal leucocytes bearing receptors for HLA molecules.     

Lack of CIITA expression is central to the absence of antigen presentation functions of trophoblast cells and is caused by methylation of the IFN-gamma inducible promoter (PIV) of CIITA

Lack of MHC-mediated antigen presenting functions of fetal trophoblast cells is an important mechanism to evade maternal immune recognition. In this study we demonstrated that the deficiency in MHC expression and antigen presentation in the trophoblast cell lines JEG-3 and JAR is caused by lack of class II transactivator (CIITA) expression due to hypermethylation of its interferon-gamma (IFN-gamma)-responsive promoter (PIV). Circumvention of this lack of CIITA expression by introduction of exogenous CIITA induced cell surface expression of HLA-DR, -DP, and -DQ, leading to an acquired capacity to present antigen to antigen-specific T cells. Transfection of CIITA in JEG-3 cells also upregulated functional HLA-B and HLA-C expression. Noteworthy, this lack of IFN-gamma-mediated induction of CIITA was also found to exist in normal trophoblast cells expanded from chorionic villus biopsies. Together, these observations demonstrate that lack of CIITA expression is central to the absence of antigen presentation functions of trophoblast cells.     

Invasion of Cytotrophoblastic (JEG-3) Cells Is Up-Regulated by Interleukin-15 In Vitro

PROBLEM: Trophoblast invasion into the uterus is controlled by many factors. Some cytokines (interleukin [IL]-1, IL-6, and IL-10) have been shown previously to play an important role in placentation. The human placenta is an important source of IL-15, although the cellular source of IL-15 in the placenta has not yet been specified. IL-15 influences cell adhesion and migration by redistributing adhesion molecules in lymphocytes and has been shown to have effects on endothelial cells and in some human tumors. METHOD OF STUDY: To study the role of IL-15 in trophoblast invasion, we investigated the effect of IL-15 (concentrations, 1–10 ng/ml) in a trophoblast invasion model (JEG-3 with matrigel-coated filters). Cell invasion was assessed using matrigel-coated filters and was expressed as the quotient of invading cells in comparison with the number of cells that had passed the control membrane. Cell migration was studied by examining the number of cells that had passed the filters without matrigel. Cell proliferation was quantified by a tetrazolium salt WST-1 cleavage assay. Matrix metalloproteinase (MMP)-1, MMP-2, and MMP-9 activities were measured by specific enzyme assays. RESULTS: IL-15 significantly (P < 0.05) increased the in vitro invasion of cytotrophoblastic (JEG-3) cells in a dose-dependent manner. There was a fourfold increase in the invasion at a concentration of 10 ng/ml of IL-15. Migration also was increased by a factor of 2.3 (P < 0.05). Cell proliferation, however, remained unchanged. The collagenolytic activity of cytotrophoblastic (JEG-3) cells was increased by IL-15 stimulation. A significant increase in MMP-1 concentration occurred after the incubation of JEG-3 cells with IL-15. No changes appeared in MMP-2, MMP-9, and tissue inhibitor of metalloproteinase-1 concentrations. CONCLUSIONS: Trophoblast invasion and migration, but not proliferation, are enhanced by IL-15. Our results suggest a role for IL-15 in the modulation of MMP-1 secretion by JEG-3 cells. Furthermore, we speculate, that IL-15 might be related to the changes of cell adhesion molecule phenotype during the process of invasion.     

Effects of Tumor Necrosis Factor-Alpha on Human Trophoblast Cell Adhesion and Motility

PROBLEM: Adhesive interaction between trophoblast cells and uterine endometrial basement membrane is one of the critical processes in embryo implantation. This interaction is directly or indirectly regulated by hormones, growth factors, and cytokines. Since tumor necrosis factor-alpha (TNF-α) is synthesized by both decidual and trophoblast cells, we hypothesized that TNF-α may play a regulatory role in trophoblast cell invasion. To test this hypothesis, we have used in vitro models to determine the effect of TNF-α on human trophoblast cell adhesion and motility, two major steps in trophoblast invasion. METHODS: The effect of TNF-α on the motility of extended-lifespan first trimester trophoblasts (HTR) and JEG-3 choriocarcinoma cells was tested using the phagokinetic track motility assay. An in vitro adhesion assay was used to determine the effect of TNF-α on the adhesion of HTR and JEG-3 cells to laminin, a major basement membrane component. In addition, the effect of TNF-α on the surface expression of the laminin receptor β1 integrin subunit was examined using flow cytometry. RESULTS: HTR or JEG-3 cells were strongly adherent to laminin which was not significantly altered by TNF-α treatment. We also measured the effect of TNF-α on the surface expression of β1 integrin on HTR and JEG-3 cells; no difference was observed between control and treatment groups. Interestingly, the motility of both HTR and choriocarcinoma JEG-3 cells was significantly inhibited by TNF-α. CONCLUSIONS: The role of TNF-α in human embryo implantation is currently unknown. Our data demonstrate that TNF-α does not alter trophoblast cell adhesion to laminin, but significantly inhibits trophoblast cell motility in vitro, suggesting that TNF-α may play a regulatory role in trophoblast cell invasion.     

HLA-G mRNA差异表达对细胞膜表面HLA-G异构体分子表达的影响

目的 探讨HLA-G异构体(HLA-G1～6)mRNA差异表达对HLA-G分子在细胞表面表达的影响.方法 通过RT-PCR方法分析卵巢癌细胞株HO-8910、H0-8910PM、OVCAR-3,白血病细胞株Jurkat、K562、HL60、MUTZ-1,绒癌细胞株JEG-3、JAR内HLA-G异构体mRNA的表达种类,采用流式细胞术分析上述细胞株细胞表面及细胞内HLA-G分子的分布及表达水平.结果 阳性对照JEG-3细胞内表达HLA-G1～6 mRNA,阴性对照JAR不表达HLA-G1～6 mRNA.HLA-G1 mRNA在HO-8910、HO-8910PM、OVCAR-3、MUTZ-1、Jurkat细胞内表达.除阳性对照JEG-3外,其他细胞均不表达HLA-G2 mRNA;表达HLA-G3 mRNA的细胞有HO-8910、HO-8910PM、K562、HL60、MUTZ-1、OVCAR-3、Jurkat;表达HLA-G4 mRNA的细胞有HO-8910、HO-8910PM、HL60、Jurkat;表达HLA-G5mRNA的细胞为Jurkat.FACS分析显示在JEG-3、HO-8910PM、Jurkat细胞膜表面表达HLA-G分子,而除JAR细胞外,其他细胞内均表达HLA-G分子.结论 HLA-G2、-G3、-G4、-G5、-G6均不能在细胞表面表达,而HIJA-G1分子则能在特定的细胞表面表达.     

二氢杨梅素对绒毛膜癌细胞增殖和迁移能力的影响

目的 探讨二氢杨梅素（DMY）对绒毛膜癌（绒癌）JEG-3及JAR细胞增殖和迁移能力的影响。 方法 MTT法检测不同浓度的二氢杨梅素(0 mg/L, 20 mg/L, 40 mg/L, 60 mg/L, 80 mg/L)作用一定时间后,对绒癌JEG-3和JAR细胞增殖能力的影响;细胞划痕实验和Transwell法检测不同浓度二氢杨梅素(0 mg/L, 40 mg/L, 60 mg/L, 80 mg/L)分别作用绒癌JEG-3细胞及JAR细胞一定时间后,对其迁移能力的影响;Real-time PCR和Western blotting方法分别检测不同浓度二氢杨梅素(0 mg/L, 40 mg/L, 60 mg/L, 80 mg/L)作用绒癌JEG-3及JAR细胞后,基质金属蛋白酶2（MMP-2）mRNA和蛋白表达水平的影响。 结果 不同浓度二氢杨梅素作用绒癌JEG-3和JAR细胞24 h和48 h后,随着二氢杨梅素浓度增加,对JEG-3和JAR细胞增殖抑制作用增强（P＜0.05）。二氢杨梅素作用绒癌JEG-3及JAR细胞后,显著抑制细胞迁移能力,且具有浓度依赖性（P＜0.05）。不同浓度二氢杨梅素作用JEG-3和JAR细胞后,MMP-2 的mRNA和蛋白表达水平明显下降（P＜0.05）。 结论 二氢杨梅素能够抑制JEG-3及JAR细胞的增殖能力且具有浓度依赖性,同时二氢杨梅素可能通过下调绒癌JEG-3及JAR细胞中MMP-2 mRNA和蛋白的表达,抑制绒癌细胞的侵袭迁移。     

Supporting the hypothesis of pregnancy as a tumor: survivin is upregulated in normal pregnant mice and participates in human trophoblast proliferation

PROBLEM: Survivin, a tumor-promoting antiapoptotic molecule, is expressed in the human placenta. Here, we analyzed its expression during normal and pathological murine pregnancy and investigated its participation in human first trimester trophoblast cell survival and proliferation. METHOD OF STUDY: We first analyzed the expression of survivin on the mRNA and protein level at the fetal-maternal interface of normal pregnant (CBA/J x BALB/c) and abortion-prone (CBA/J x DBA/2J) mice at different pregnancy stages by RT-PCR and immunohistochemistry. We also evaluated apoptosis in murine trophoblasts in both mating combinations by TUNEL technique. Functional studies were carried out by knockdown survivin by means of siRNA methodology in two human first trimester trophoblast cell lines [Swan.71 (Sw.71) and HTR8 (H8)]. RESULTS: We observed a peak in mRNA levels on day 5 and a peak of protein levels on day 8 of pregnancy in both combinations. The level of survivin in animals from the abortion-prone group was decreased compared with normal pregnant mice on day 8, which was accompanied by elevated apoptosis rates. In later pregnancy stages (days 10 and 14), survivin levels decreased to levels comparable to those observed right after fecundation in both groups. Transfection of human first trimester cell lines (H8 and Sw.71) with siRNA targeting the survivin gene led to a 76-82% reduction of its expression leading to reduced trophoblast cell viability and proliferation. CONCLUSION: Our findings suggest an important role of survivin to promote trophoblast cell survival and proliferation during placentation, thus maintaining pregnancy. The pregnancy-associated expression of a cancer molecule such as survivin supports the 'pseudo-malignancy' hypothesis of pregnancy. Our data may contribute to the better understanding of trophoblast cell development during implantation and placentation.     

Corticotropin-releasing hormone modulates human trophoblast invasion through carcinoembryonic antigen-related cell adhesion molecule-1 regulation

Abnormalities in the process of trophoblast invasion may result in abnormal placentation. Both the embryonic trophoblast and maternal decidua produce corticotropin-releasing hormone (CRH), which promotes implantation. Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), which is expressed in extravillous trophoblasts (EVTs) of normal human placenta, may also function in tro-phoblast/endometrial interactions. We investigated whether locally produced CRH plays a role in trophoblast invasion, primarily by regulating CEACAM1 expression. We examined cultures of freshly isolated human EVTs, which express CEACAM1, and an EVT-based hybridoma cell line, which is devoid of endogenous CEACAM1. CRH inhibited EVT invasion in Matrigel invasion assays, and this effect was blocked by the CRH receptor type 1 (CRHR1)-specific antagonist antalarmin. Additionally, CRH decreased CEACAM1 expression in EVTs in a dose-dependent manner. After transfection of the hybridoma cell line with a CEACAM1 expression vector, the invasiveness of these cells was strongly enhanced. This effect was inhibited by addition of blocking monoclonal antibody against CEACAM1. Furthermore, blocking of endogenous CEACAM1 in EVTs inhibited the invasive potential of these cells. Taken together these findings suggest that CRH inhibits trophoblast invasion by decreasing the expression of CEACAM1 through CRHR1, an effect that might be involved in the pathophysiology of clinical conditions, such as preeclampsia and placenta accreta.     

Distribution patterns of matrix metalloproteinase (MMP)-2 and -9 and their inhibitors (TIMP-1 and TIMP-2) in the human decidua during early pregnancy

Early human trophoblast shows dramatic invasive properties during early pregnancy. A tightly-regulated activation of matrix metalloproteinases (MMPs) is considered to be of critical importance for the control of trophoblast invasion. The aim of the present study was to determine MMP-2, MMP-9, TIMP-1 and TIMP-2 protein expression in decidual endometrium during the first trimester of pregnancy (22-42 days post coitus) with special attention to their expression patterns in endometrial compartments. Cytokeratin staining applied to adjacent sections was used to identify epithelial and trophoblast cells. We observed that MMP-2, particularly in the fourth week, appeared to be expressed more strongly in extravillous trophoblasts (EVTs) and vascular endothelial cells in the first trimester of pregnancy. Therefore, MMP-2 is likely to be the primary mediator in invasion of the trophoblast into the decidual endometrium, as well as in vascular remodeling and angiogenesis in the first trimester of pregnancy. The high expression of TIMP-1 and TIMP-2 in EVTs and glandular epithelium suggests that a restricted and balanced expression of these molecules is important for matrix remodeling and controlled trophoblast invasion during placentation. We conclude that (1) MMP-2 and MMP-9 and their inhibitors TIMP-1, and TIMP-2 determine the invasive behavior of trophoblast into the endometrium, and in particular, (2) MMP-2 may be the key regulator of trophoblast invasion in early human pregnancy.