Treatment Of JEG-3 Cells Research Articles

CYP19A1 Expression Is Controlled by mRNA Stability of the Upstream Transcription Factor AP-2γ in Placental JEG3 Cells.

CYP19A1 encodes aromatase, which converts testosterone to estrogen, and is induced during placental maturation. To elucidate the molecular mechanism underlying this function, histone methylation was analyzed using the placental cytotrophoblast cell line, JEG3. Treatment of JEG3 cells with 3-deazaneplanocin A, an inhibitor of several methyltransferases, resulted in increased CYP19A1 expression, accompanied by removal of the repressive mark H3K27me3 from the CYP19A1 promoter. However, this increase was not observed in cells treated with GSK126, another specific inhibitor for H3K27me3 methylation. Expression of TFAP2C, which encodes AP-2γ, a transcription factor that regulates CYP19A1, was also elevated on 3-deazaneplanocin A treatment. Interestingly, TFAP2C messenger RNA (mRNA) was readily degraded in JEG3 cells but protected from degradation in the presence of 3-deazaneplanocin A. TFAP2C mRNA contained N6-methyladenosines, which were reduced on drug treatment. These observations indicate that the TFAP2C mRNA undergoes adenosine methylation and rapid degradation, whereas 3-deazaneplanocin A suppresses methylation, resulting in an increase in AP-2γ levels. We conclude that the increase in AP-2γ expression via stabilization of the TFAP2C mRNA is likely to underlie the increased CYP19A1 expression.

Dexamethasone may inhibit placental growth by blocking glucocorticoid receptors via phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin and reactive oxygen species/AMP-activated protein kinase signalling pathways in human placental JEG-3 cells.

This study explored the molecular mechanism underlying the effects of dexamethasone (DEX, 1 µM) on glucose transporters (GLUT) in JEG-3 human placental choriocarcinoma cells. JEG-3 cells were treated with DEX, an expression plasmid encoding human glucocorticoid receptor α (GRα), pcDNA3.1-GRα, GRα short interference (si) RNA, LY294002, xanthine oxidase (XO)/hypoxanthine (HX), rapamycin, insulin-like growth factor (IGF) 1, N-acetylcysteine (NAC) or phosphatidic acid (PA), and cell proliferation, apoptosis, mitochondrial membrane potential (MMP), human chorionic gonadotrophin (hCG) content, human placental lactogen (hPL) content, glucose uptake, reactive oxygen species levels and signalling pathway modulation were evaluated. Treatment of JEG-3 cells with DEX (1 µM), GRα siRNA, LY294002 (50 µM), XO/HX (7.2 µM/36 nM) or rapamycin (80 nM) inhibited cell proliferation, induced apoptosis, significantly decreased MMP and hCG and hPL content and increased ROS levels. In addition, glucose uptake was decreased through downregulation of the mRNA and protein expression of GRα, GLUT1 and GLUT3. Treatment of JEG-3 cells with GRα siRNA, LY294002, XO/HX or rapamycin inhibited phosphorylation of phosphatidylinositol 3-kinase (PI3K), Akt, glycogen synthase kinase 3 and mammalian target of rapamycin (mTOR) and induced the phosphorylation of AMP-activated protein kinase (AMPK) and tuberous sclerosis complex 2. The effects of GRα overexpression and IGF1 (100 nM), NAC (5 nM) or PA (100 µM) treatment on JEG-3 cells contrasted with those of DEX treatment. DEX blocked glucose uptake by downregulating GRα expression, which reduced GLUT1 and GLUT3 mRNA and protein expression, which, in turn, may have inhibited the PI3K/AKT/mTOR pathway and activated the ROS/AMPK pathway.

The von Hippel Lindau tumour suppressor gene is a novel target of E2F4-mediated transcriptional repression in preeclampsia

The von Hippel Lindau tumour suppressor (VHL) protein is essential for proper placental development and is downregulated in preeclampsia (PE), a devastating disorder of pregnancy typified by chronic hypoxia. To date, knowledge on VHL genetic and epigenetic regulation is restricted to inactivating mutations and loss-of-heterozygosity in renal cell carcinomas. Herein, we sought to examine whether VHL DNA is subject to differential methylation, and if so, whether it is altered in early-onset PE (E-PE). Sodium bisulfite modification and methylation-specific PCR analysis revealed that VHL is subject to extensive methylation in a CpG-rich region within its promoter in the human placenta. Notably, we detected significant differences in methylation in E-PE placentae relative to normotensive age-matched controls at key transcription factor binding sites, including that of the transcriptional repressor E2F4. Treatment of JEG3 cells with 5-Aza-2′-deoxycytidine, revealed that loss of DNA methylation promoted VHL transcription by attenuating VHL association with E2F4. RNAi knockdown of E2F4 in vitro confirmed its function on VHL repression. Exposure of JEG3 cells to transforming growth factor beta (TGFβ) downregulated VHL mRNA. In line with elevated levels of TGFβ3 in E-PE, chromatin immunoprecipitation assays revealed that E2F4-VHL association was enhanced upon TGFβ3 treatment, indicative of VHL transcriptional inhibition. In line with decreased VHL expression in E-PE, augmented E2F4-VHL association was also observed in E-PE placental tissue relative to controls. In conclusion, we demonstrate for the first time that hypomethylation of VHL DNA at a key transcription factor binding site has significant consequences for its transcriptional repression in early-onset preeclampsia.

Placental autophagy regulation by the BOK-MCL1 rheostat

Autophagy is the catabolic degradation of cellular cytoplasmic constituents via the lysosomal pathway that physiologically elicits a primarily cytoprotective function, but can rapidly be upregulated in response to stressors thereby inducing cell death. We have reported that the balance between the BCL2 family proteins BOK and MCL1 regulates human trophoblast cell fate and its alteration toward cell death typifies preeclampsia. Here we demonstrate that BOK is a potent inducer of autophagy as shown by increased LC3B-II production, autophagosomal formation and lysosomal activation in HEK 293. In contrast, using JEG3 cells we showed that prosurvival MCL1 acts as a repressor of autophagy via an interaction with BECN1, which is abrogated by BOK. We found that MCL1-cleaved products, specifically MCL1c157, trigger autophagy while the splicing variant MCL1S has no effect. Treatment of JEG3 cells with nitric oxide donor SNP resulted in BOK-MCL1 rheostat dysregulation, favoring BOK accumulation, thereby inducing autophagy. Overexpression of MCL1 rescued oxidative stress-induced autophagy. Of clinical relevance, we report aberrant autophagy levels in the preeclamptic placenta due to impaired recruitment of BECN1 to MCL1. Our data provided the first evidence for a key role of the BOK-MCL1 system in regulating autophagy in the human placenta, whereby an adverse environment as seen in preeclampsia tilts the BOK-MCL1 balance toward the build-up of isoforms that triggers placental autophagy.

Placental DEPTOR as a stress sensor during pregnancy

DEPTOR [DEP-domain-containing and mTOR (mammalian target of rapamycin)-interacting protein] is a modulator of mTOR signalling that binds to mTORC (mTOR complex) 1 and mTORC2. However, to date, the precise functions of DEPTOR are not fully elucidated, particularly in reproductive tissues where mTOR acts as a placental nutrient sensor. Pregnancy is associated with major physiological and psychosocial changes and adaptation to these changes is crucial for normal fetal development. In the present study, we tested the hypothesis that maternal stress can affect mTOR signalling at term, and, as a result, influence placental growth. We first investigated the expression of DEPTOR, mTOR, rictor (rapamycin-insensitive companion of mTOR) and raptor (regulatory associated protein of mTOR) from human placentas (n=23) using Q-PCR (quantitative PCR), and correlated these data to days of pregnancy and maternal stress, as well as placental and fetal weight. Maternal and fetal cortisol levels were also measured. JEG-3 and BeWo cells, used as placental in vitro models, were treated with cortisol and DEPTOR expression was assessed using Q-PCR. DEPTOR appears to be the predominant transcript in the human placenta compared with mTOR, rictor and raptor in both term (n=13) and preterm (n=10) placentas as assessed by Q-PCR. There was a significantly lower level only of log-DEPTOR gene expression in the high stress group (−1.34) than in the low stress group (0.07; t20=2.41, P=0.026). Interestingly, mothers with high stress had significantly elevated levels of cortisol (8555 pg/ml) compared with those with low stress (4900 pg/ml). We then tested the hypothesis that cortisol can directly affect DEPTOR expression. When BeWo cells were treated with cortisol 10, 100 and 1000 nM, the expression of DEPTOR was significantly down-regulated by 50, 41 and 39% (all P<0.05) respectively when compared with basal levels. Treatment of JEG-3 cells with cortisol, led to a significant decrease of DEPTOR expression at 100 nM (39%, P<0.05) and at 1000 nM (73%, P<0.01). These novel findings are indicative of a higher order of complexity of DEPTOR signalling in the human placenta that is affected by maternal stress, which could affect pregnancy outcome.

Interleukin-11 increases invasiveness of JEG-3 choriocarcinoma cells by modulating STAT3 expression

Amongst the interleukin-6 (IL-6) family of cytokines, leukemia inhibitory factor (LIF) has been shown to promote trophoblast invasion and proliferation. In the present study interleukin-11 (IL-11), another member of the IL-6 family, was investigated for its role in regulating invasion, migration and proliferation of JEG-3 choriocarcinoma cells. JEG-3 cells, like extra villous trophoblast (EVT), express mRNA transcripts encoding IL-11 and IL-11 receptor-alpha (IL-11Ralpha). Treatment of JEG-3 cells with IL-11 led to an increase in invasion across Matrigel extracellular matrix without an increase in proliferation. There was a dose-dependent increase in activation of STAT3 under the influence of IL-11 with maximum Tyr705 phosphorylation by 10min. In addition, treatment of JEG-3 cells with IL-11 for 24h led to an increase in expression of unphosphorylated STAT1 and STAT3. Analysis of the nuclear fraction showed an increased localization of STAT3 following IL-11 treatment while STAT1 was absent. Silencing the expression of STAT3 by siRNA caused a 25% reduction in invasion compared to control cells, however this was not significant. Furthermore, treatment of STAT3-silenced JEG-3 cells with IL-11 led to a significant increase in invasion compared to STAT3-silenced cells without cytokine, but this was not significant compared to non-transfected control cells. Silencing the expression of gp130 but not of IL-6R abrogated the increase in invasiveness of JEG-3 cells following IL-11 treatment. In conclusion, activation and upregulation of STAT3 appears to be critical for the IL-11-mediated increase in invasiveness of JEG-3 cells.

A protein kinase C-independent pathway leading to c-Jun-dependent expression of 100-kDa Ras GTPase-activating protein in JEG-3 human choriocarcinoma cells.

Although the 100-kDa Ras GTPase-activating protein (p100 RasGAP) has been reported to exist specifically in human placental trophoblasts, the molecular mechanisms responsible for regulating its expression remain unclear. In this study we used okadaic acid, an inhibitor of serine/threonine phosphatase 1 and 2 A, as a probe to explore the signaling pathway regulating the expression of p100 RasGAP in JEG-3 human placental choriocarcinoma cells. Treatment of JEG-3 cells with okadaic acid provoked dose- and time-dependent stimulation of p100 RasGAP expression without marked modification of expression of p120 RasGAP, another isoform of RasGAP. Co-treatment of cells with okadaic acid and the protein kinase C activator, phorbol 12-myristate 13-acetate, exerted an additive effect on p100 RasGAP induction. Moreover, the response of the p100 RasGAP de novo synthesis to okadaic acid was not affected by the selective inhibitor of protein kinase C, GF 109203X. Thus this study identified a novel signaling pathway regulating p100 RasGAP expression, which is independent of protein kinase C. In addition, okadaic acid treatment resulted in the activation of ERK2 (p42 MAP kinase) and the induction of both c-Jun and c-Fos proteins without activating JNK (c-Jun NH2-terminal kinase). Significantly, blockade of c-Jun expression with antisense c-jun oligonucleotides suppressed p100 RasGAP expression. Taken together, it is concluded that okadaic acid induces the expression of p100 RasGAP protein in JEG-3 cells preceded by activation of ERK and AP-1 cascade, and that this okadaic acid-induced p100 RasGAP expression is independent of protein kinase C-mediated pathway but requires c-Jun/AP-1 function.

Search for a human homologue of the mouse Ped gene.

The Ped gene influences the rate of cleavage division of preimplantation mouse embryos and subsequent embryonic survival. The mouse Ped gene product is a major histocompatibility complex (MHC) class Ib protein called Qa-2. Studies from many human in-vitro fertilization (IVF) clinics suggest that the mouse Ped gene has a human homologue because embryos fertilized at the same time have different cleavage rates, and those embryos that cleave at a faster rate are more likely to result in a viable pregnancy. Candidates for the human homologue of the mouse Ped gene include the MHC class Ib genes HLA-E, HLA-F, and HLA-G. The presence of mRNA for these three genes was tested in 108 spare day 3 human preimplantation embryos from 25 couples by using reverse transcription-polymerase chain reaction (RT-PCR). Of the 86 embryos tested for HLA-E mRNA, 72 were positive (84%), and of the 88 embryos tested for HLA-G mRNA, 39 were positive (44%). None of the 17 embryos tested for HLA-F mRNA were positive (0%). Studies of expression of HLA-G protein were undertaken to ascertain whether HLA-G was attached to the cell membrane via a glycosylphosphatidylinositol (GPI) linkage similar to that found in Qa-2 protein. Treatment of JEG-3 cells, an HLA-G expressing cell line, with phospholipase C did not result in removal of HLA-G showing that HLA-G, unlike Qa-2, is not GPI linked to the cell surface. The pros and cons of HLA-E, HLA-F, and HLA-G as candidates for the human Ped gene are discussed.

Identification of trophoblast in the peripheral blood by expression of mRNA of human chorionic gonadotropin (hCG) β -subunit

Amongst the interleukin-6 (IL-6) family of cytokines, leukemia inhibitory factor (LIF) has been shown to promote trophoblast invasion and proliferation. In the present study interleukin-11 (IL-11), another member of the IL-6 family, was investigated for its role in regulating invasion, migration and proliferation of JEG-3 choriocarcinoma cells. JEG-3 cells, like extra villous trophoblast (EVT), express mRNA transcripts encoding IL-11 and IL-11 receptor-α (IL-11Rα). Treatment of JEG-3 cells with IL-11 led to an increase in invasion across Matrigel extracellular matrix without an increase in proliferation. There was a dose-dependent increase in activation of STAT3 under the influence of IL-11 with maximum Tyr705 phosphorylation by 10 min. In addition, treatment of JEG-3 cells with IL-11 for 24 h led to an increase in expression of unphosphorylated STAT1 and STAT3. Analysis of the nuclear fraction showed an increased localization of STAT3 following IL-11 treatment while STAT1 was absent. Silencing the expression of STAT3 by siRNA caused a 25% reduction in invasion compared to control cells, however this was not significant. Furthermore, treatment of STAT3-silenced JEG-3 cells with IL-11 led to a significant increase in invasion compared to STAT3-silenced cells without cytokine, but this was not significant compared to non-transfected control cells. Silencing the expression of gp130 but not of IL-6R abrogated the increase in invasiveness of JEG-3 cells following IL-11 treatment. In conclusion, activation and upregulation of STAT3 appears to be critical for the IL-11-mediated increase in invasiveness of JEG-3 cells.

Hypoxia downregulates continuous and interleukin-1-induced expression of human chorionic gonadotropin in choriocarcinoma cells

The effects of hypoxia on JEG-3, BeWo, and JAr cells were investigated and it was demonstrated that choriocarcinoma cells can be used as a model to study the molecular mechanism of hypoxia-mediated repression of human chorionic gonadotropin (hCG). Cells were maintained under hypoxia (3.5 per cent OZ) for 72 h without loss of viability, as demonstrated by the fact that 93–98 per cent of the cells excluded trypan blue. Up to 48 h, cell growth was not significantly influenced by hypoxia, and analysis by flow cytometry did not reveal major changes in cell cycle distribution. JEG-3, BeWo, and JAr cells which were grown for 48 h under hypoxia secreted 81, 67, and 71 per cent less hCG than cells cultivated under normoxic conditions. The extent of hCG reduction was dependent on the oxygen concentration. Moreover, release of the hormone from hypoxic JAr cells was not stimulated upon addition of interleukin-1 (IL-1). Treatment of JEG-3 cells with methotrexate (MTX) led to a 4.3-fold augmentation in hCG secretion and to an increase in the amount of G 0G 11 cells. However, when cells were cultured in the presence of MTX and hypoxia, hCG secretion decreased 10-fold and βhCG mRNA declined to almost undetectable levels suggesting that downregulation of βhCG mRNA is the major cause of diminished hCG release under hypoxic conditions.

Ethanol and mitotic inhibitors promote differentiation of trophoblastic cells.

Chronic ethanol abuse during pregnancy can cause fetal injury. A contributing factor in this fetal injury may be the effect of ethanol on the placenta. Ethanol treatment increases human chorionic gonadotropin (hCG) production by cultured human placental trophoblasts. In this study, we show that ethanol treatment reduces total DNA and total protein while stimulating hCG production in term trophoblasts. Ethanol treatment inhibits growth in rapidly proliferating trophoblastic cells from a first trimester placenta and JEG-3 choriocarcinoma cells. In both cell types, the normal increases in total DNA were inhibited in an ethanol dose-dependent manner. Normal increases in total protein were inhibited as well. In contrast, hCG production, an indicator of differentiation, was stimulated by ethanol treatment. Treatment of JEG-3 cells with antimitogenic agents, methotrexate (MTX) or cytosine arabinoside (Ara-C), inhibited cell growth as indicated by decreased total DNA and total protein accumulation. Similar to that with ethanol treatment, inhibition of cell proliferation was accompanied by increases in hCG production. Taken together, these data suggest that one mechanism by which ethanol increases hCG production in human placental trophoblasts may involve alterations in cellular growth and/or differentiation; such alterations may also occur in other proliferating cells in the growing fetus.

An immuno suppressive inducer factor secreted by JEG 3 choriocarcinoma cell line: In vitro and in vivo studies

Expression of the c-myc proto-oncogene has been shown to correlate with the rate of cellular proliferation and malignant transformation in a number of cell types. JEG-3 choriocarcinoma cells demonstrate c-myc transcript levels that are greater than those of nonmalignant trophoblastic tissue at any stage of gestation. Southern blot analysis documents c-myc gene amplification in JEG 3 cells, with a gene copy number of approximately 20. The methylation pattern and genomic structure of the amplified c-myc oncogene in JEG-3 choriocaricoma cells are identical to those of normal placenta. Treatment of JEG-3 cells with mithramycin, a ribonucleic acid synthesis inhibitor, results in a dramatic decrease in c-myc expression relative to that of the c-Ha-Ras gene. The apparent selectivity of mithramycin for c-myc expression represents the only example, to date, of the selective pharmacologic modulation of oncogene expression.

Differentiation-dependent expression of the BCL-2 proto-oncogene in the human trophoblast lineage.

We explored the role of the BCL-2 proto-oncogene in the life cycle of trophoblast cells by examining: 1) the patterns of BCL-2 expression in normal placenta at various gestational ages and in specimens of hydatidiform moles and choriocarcinomas, and 2) the effects of cyclic adenosine monophosphate (cAMP) treatment of JEG-3 choriocarcinoma cells, which induces differentiated functions, on BCL-2. BCL-2 protein was localized by indirect immunofluorescence and immunoperoxidase staining of tissue sections and cells using monoclonal and polyclonal antibodies. Western and Northern blotting were used to assess BCL-2 and p53 protein and mRNA levels, respectively. JEG-3 cells were transfected with a BCL-2 expression plasmid to establish that BCL-2 protein could be expressed at high levels in this cell type. BCL-2 immunostaining was most prominent in the syncytiotrophoblast of normal placenta. It was found in syncytiotrophoblast of complete and partial hydatidiform moles, whereas cytotrophoblast staining was weak. BCL-2 immunostaining was also barely detectable in choriocarcinoma cells (JEG-3 cells) and a primary choriocarcinoma. However, BCL-2 protein could be transiently overexpressed in JEG-3 cells by transfection with an expression plasmid. Western blot analysis revealed low levels of BCL-2 in JEG-3 cells and a rise in BCL-2 protein in placental extracts from 10 weeks' gestation to term. In contrast, p53 protein was abundant in JEG-3 cells and normal placenta at 10 weeks' gestation, but low at term, BCL-2 transcripts were substantially more abundant in term placenta than in JEG-3 cells. Treatment of JEG-3 cells with 8-Br-cAMP, which induces genes characteristic of the syncytiotrophoblast, raised BCL-2 protein approximately twofold, whereas p53 mRNA declined. We conclude that: 1) There is a differentiation-dependent pattern of BCL-2 expression in the placenta, with the protein being most abundant in terminally differentiated trophoblast cells; 2) there appears to be an inverse relation between BCL-2 and p53 expression in trophoblast; and 3) cAMP regulates BCL-2 protein in trophoblast cells. We speculate that the expression of BCL-2 in terminally differentiated trophoblast cells, and hence resistance to apoptotic cell death, may be one mechanism by which trophoblast mass is preserved during pregnancy. Conversely, the relatively low expression of BCL-2 in choriocarcinoma cells may render them more susceptible to apoptosis.

Selective inhibition of c-myc expression by the ribonucleic acid synthesis inhibitor mithramycin

Expression of the c-myc proto-oncogene has been shown to correlate with the rate of cellular proliferation and malignant transformation in a number of cell types. JEG-3 choriocarcinoma cells demonstrate c-myc transcript levels that are greater than those of nonmalignant trophoblastic tissue at any stage of gestation. Southern blot analysis documents c-myc gene amplification in JEG 3 cells, with a gene copy number of approximately 20. The methylation pattern and genomic structure of the amplified c-myc oncogene in JEG-3 choriocaricoma cells are identical to those of normal placenta. Treatment of JEG-3 cells with mithramycin, a ribonucleic acid synthesis inhibitor, results in a dramatic decrease in c-myc expression relative to that of the c-Ha-Ras gene. The apparent selectivity of mithramycin for c-myc expression represents the only example, to date, of the selective pharmacologic modulation of oncogene expression.