Serum-dependent effects of IGF-I and insulin on proliferation and invasion of human first trimester trophoblast cell models

Extravillous cytotrophoblasts are specialised epithelial cells of the placenta that proliferate or invade the maternal decidua. Little is known about the mechanisms that regulate these processes. Here the effects of several insulin and insulin-like growth factor-I (IGF-I) doses, either singly or in synergy with serum, on human chorionic gonadotropin-beta (hCG-beta) secretion (RIA), proliferation (cell counting, cyclin B(1) levels) and invasion [Matrigel invasion assay, secretion of matrix metalloproteinases (MMP) 2 and 9] were investigated. The choriocarcinoma cell lines BeWo, JAR and JEG-3 served as models for first trimester human trophoblasts. Both growth factors altered hCG-beta secretion and proliferation dependent on the cell line. Insulin stimulated proliferation in JAR cells and, to a lesser extent, in JEG-3 cells, and when cultured in serum-free medium, BeWo was not affected. Invasion was not affected although proMMP-2 levels in culture medium were altered under some conditions. A strong synergistic effect with serum was noted. In the presence of serum both growth factors reduced proliferation and invasion in a similar fashion. Since the cell models differ by their degree of differentiation, the data demonstrate that the effects of insulin and IGF-I strongly depend on serum and the degree of differentiation. It can be speculated that IGF-I can take on tasks of insulin in the regulation of trophoblast functions under conditions of insulinopenia.     

HCG-Activated Human Peripheral Blood Mononuclear Cells (PBMC) Promote Trophoblast Cell Invasion

Successful embryo implantation and placentation depend on appropriate trophoblast invasion into the maternal endometrial stroma. Human chorionic gonadotropin (hCG) is one of the earliest embryo-derived secreted signals in the peripheral blood mononuclear cells (PBMC) that abundantly expresses hCG receptors. The aims of this study were to estimate the effect of human embryo–secreted hCG on PBMC function and investigate the role and underlying mechanisms of activated PBMC in trophoblast invasion. Blood samples were collected from women undergoing benign gynecological surgery during the mid-secretory phase. PBMC were isolated and stimulated with or without hCG for 0 or 24 h. Interleukin-1β (IL-1β) and leukemia inhibitory factor (LIF) expressions in PBMC were detected by enzyme-linked immunosorbent assay and real-time polymerase chain reaction (PCR). The JAR cell line served as a model for trophoblast cells and was divided into four groups: control, hCG only, PBMC only, and PBMC with hCG. JAR cell invasive and proliferative abilities were detected by trans-well and CCK8 assays and matrix metalloproteinase (MMP)-2 (MMP-2), MMP-9, vascular endothelial growth factor (VEGF), tissue inhibitor of metalloproteinase (TIMP)-1, and TIMP-2 expressions in JAR cells were detected by western blotting and real-time PCR analysis. We found that hCG can remarkably promote IL-1β and LIF promotion in PBMC after 24-h culture. PBMC activated by hCG significantly increased the number of invasive JAR cells in an invasion assay without affecting proliferation, and hCG-activated PBMC significantly increased MMP-2, MMP-9, and VEGF and decreased TIMP-1 and TIMP-2 expressions in JAR cells in a dose-dependent manner. This study demonstrated that hCG stimulates cytokine secretion in human PBMC and could stimulate trophoblast invasion.     

Effect of human parathyroid hormone on the cAMP production and the endocrine functions of trophoblast cells from first trimester placenta.

Our previous study on teratocarcinoma cells suggested the role of human parathyroid hormone (hPTH) in early development of the placenta. The purpose of this study was to evaluate the possible role of hPTH on the functions of first trimester trophoblast cells. Adenylate cyclase activity in crude membranes from first trimester human placental villous tissue is stimulated 2-fold by hPTH (1-34) (10(-6) mol.l-1) from 265 +/- 32 to 532 +/- 80 pmol of cAMP/mg protein/15 min. A similar stimulation of adenylate cyclase is observed in human term placental villous tissue but not in 3 different choriocarcinoma cell lines. In order to evaluate the possible role of hPTH on the functions of first trimester human trophoblast cells, these cells were isolated by dispase and cultured (2 x 10(5) cells per plate) in DMEM supplemented with 20% fetal calf serum with or without 100 ng/ml of epidermal growth factor (EGF), for 4 d. On d 2 of culture, hPTH (10(-7) mol.l-1) stimulates cAMP production of these cells from 0.52 +/- 0.2 to 2.58 +/- 0.57 pmol.h-1 per 10(6) cells (mean +/- SEM). As compared to control (30 ng/ml), the output of hCG is increased by 1.5- (NS), 2- (P less than 0.01) and 3- (P less than 0.01) fold by EGF, hPTH, and hPTH added with EGF, respectively. Dibutyryl cAMP (10(-3) mol.l-1) increased hCG secretion by 3-fold (P less than 0.05). EGF and hPTH added separately or together significantly stimulated (P less than 0.01) the secretion of free alpha subunit 2-fold from 35 ng/ml to 70 ng/ml. In contrast, hPTH and EGF added separately did not change the secretion of free beta hCG. However, added together, they significantly increased (P less than 0.01) the secretion of free beta hCG after 48 h of culture, maximal stimulation (2.5 fold) being observed at d 4 of culture. In conclusion, human trophoblast cells are target cells for hPTH. hPTH acts in association with EGF in promoting expression of endocrine activity of these cells, such as hCG secretion. Trophoblast cells provide a model for the study of the cooperative effect between a peptide hormone and a growth factor in the regulation of endocrine function.     

The use of drugs to dissect the pathway for secretion of the glycoprotein hormone chorionic gonadotropin by cultured human trophoblastic cells

Agents that affect intracellular cation and pH gradients and inhibit energy production have been tested for their ability to modulate the processing and secretion of the free alpha subunit and the alpha beta dimer of human chorionic gonadotropin (hCG) by cultured human trophoblastic cells (JAR). Incubation of JAR cells with monensin or nigericin, monovalent cation ionophores that produce equilibration of Na+ and K+ across cellular membranes, dicyclohexylcarbodiimide, an agent that inhibits intracellular membrane ATPases, and methylamine, which neutralizes intracellular pH gradients, produced similar effects on hCG processing and secretion. All these agents inhibited the processing of the asparagine-linked oligosaccharide chains of free alpha subunit and the alpha and beta subunits contained in the hCG dimer. Moreover, after treatment of JAR cells with these agents, there was an intracellular accumulation of precursor forms and an inhibition of secretion of "mature" forms of hCG. Monensin affected the processing and secretion of hCG subunits differently at different concentrations. At 5 X 10(-7) M, monensin inhibited the processing of the asparagine-linked oligosaccharides of hCG without altering the rate-limiting step in the secretory pathway or blocking hCG secretion. The intracellular hCG subunit precursors in both control and monensin-treated cells contained a similar array of high mannose oligosaccharides, predominantly of the Man8GlcNAc2 and Man9GlcNAc2 types. However, monensin-treated cells secreted hCG subunits that contained endo H-sensitive oligosaccharides of the high mannose (mostly Man5GlcNAc2) and hybrid types rather than the endo H-resistant complex chains synthesized by control cells. Nevertheless, a full complement of serine-linked oligosaccharides was added to the hCG-beta subunit in monensin-treated cells. These results indicate that the intracellular movement of hCG from the rough endoplasmic reticulum to the cell surface was not inhibited by monensin at a concentration that impaired Golgi-localized steps in the processing of asparagine-linked oligosaccharides. At 5 X 10(-6) M, monensin significantly inhibited secretion of hCG and created a new rate-limiting step in the processing pathway. hCG subunits bearing Man5GlcNAc2 units accumulated intracellularly, suggesting that the equilibration of intracellular Na+/K+ pools blocked oligosaccharide processing at an intra-Golgi point, perhaps by inhibiting movement of the glycoprotein hormone from the "cis" to the "trans" Golgi compartment. Since the other drugs mentioned above produced similar effects on hCG processing and secretion, it appears that maintenance of intracellular cation and pH gradients is necessary for the intra-Golgi transport of glycoprotein hormones.(ABSTRACT TRUNCATED AT 400 WORDS)     

In vitro evaluation of combination of EGCG and Erlotinib with classical chemotherapeutics on JAR cells

Gestational Trophoblastic Neoplasia (GTN) is a term used for a group of malignant gynecological tumors including choriocarcinoma. Low-risk neoplasias can be cured using single agents Methotrexate (MTX) and actinomycin-D (ACD), but in certain cases, decreased responsiveness and serious side effects occur. Therefore, researchers have been attempting to find new treatment modalities. One of the most popular way for increasing cancer patient survival rates is supporting treatment with adjuvant molecules or chemosensitizers. For this purpose, we investigated epigallocatechin-3-gallate (EGCG), a green tea cathecin, and Erlotinib, an EGFR tyrosine kinase inhibitor, as single agents and combined with MTX or ACD. In accordance with this, JAR (human placenta choriocarcinoma) cell line was used as an in vitro model and MTT, LDH, caspase-3 activation, RT-PCR, and Western Blot analyses were performed to investigate the effects of the test materials. Our studies demonstrate that combination of Erlotinib and EGCG with MTX and ACD decreases JAR cell proliferation and metastatic HER2 protein synthesis and increases caspase-3 activation compared to ACD or MTX alone. In addition, significant increase was observed in the apoptotic Bax gene, but no notable protein synthesis occurred in the Western Blot analysis, which suggests that combination of Erlotinib and EGCG with classical chemotherapeutics ACD or MTX may lead the JAR cells to apoptosis, but not by a mitochondrial pathway. All the results indicate that the synergetic effect of Erlotinib and EGCG with classical chemotherapeutics may help to increase patient survival rates of choriocarcinoma, but the detailed mechanism needs further investigation.     

Self-regulation of the endothelin receptor system in choriocarcinoma cells

The human trophoblast secretes endothelin-1 (ET-1) and expresses ET receptors. The present study tested whether the transformed BeWo, JAR and JEG-3 choriocarcinoma cells: (1) secrete endothelin-1 (ET-1); (2) express both ET-A and ET-B receptor subtypes; and (3) have the potential to allow for autologous regulation of ET-receptor proteins. The cells were cultured for 24/48 h with or without 10% FCS and, in experiments on receptor regulation, with ET-1 (5-20 nM and 10 microM). ET-1 secretion was measured by RIA and receptor levels by immunoblotting. All cell types secreted ET-1 albeit at different levels and sensitivity to FCS. All cell lines expressed both ET-A (JEG-3>BeWo=JAR) and ET-B (JEG-3=JAR>BeWo) receptor subtypes, which could be up- and downregulated depending on ET-1 concentration, culture time and FCS presence. It is concluded that BeWo, JAR and JEG-3 choriocarcinoma cells secrete ET-1 and express both ET-A and ET-B receptor subtypes. The receptor levels can be regulated by ET-1. This provides the molecular basis for an autocrine system with the potential of autologous regulation of yet unidentified ET-1-induced functions.     

Failure of contraceptive steroids to modify human chorionic gonadotrophin secretion by hydatidiform mole tissue and choriocarcinoma cells in culture

The effect of steroids contained in oral contraceptives, namely ethinylestradiol:17α-ethinyl-1,3,5(10)-estratriene-3, 17-diol (E) and norethindrone acetate: 17β-acetoxy-17-ethinyl-4-estren-3-one (N), on cell replication and human chorionic gonadotropin (hCG) secretion by choriocarcinoma cells in monolayer culture and by hydatidiform mole tissue maintained in organ culture were studied. The steroids were added to the culture medium individually or in combination to achieve a range of concentrations (10^?10 to 10^?4), within and beyond the presumed concentration of these substances in the blood of women taking oral contraceptives. The effect of luteinizing hormone releasing hormone (LHRH) on hCG secretion by choriocarcinoma cells in monolayer culture also was investigated. The rate of hCG production by either choriocarcinoma cells in monolayer culture or by hydatidiform mole tissue maintained in organ culture was not affected by the hormones used in this study; indeed hCG secretion remained reasonably unchanged even with high concentrations of steroids (up to 10^?14 M) or LHRH (up to 10^?4 mg × ml^?1). Cell replication, as measured by increase in amount of cellular protein and DNA, was not stimulated by either of these compounds.     

Amphiregulin regulates the production of human chorionic gonadotropin in trophoblasts

AimsThe aim of this study was to investigate the significance of epidermal growth factor receptor (EGFR) ligands produced in syncytiotrophoblasts during normal pregnancy.Main methodsWe examined the expression of EGFR ligands in human pregnancy by real-time PCR, and analyzed the relationship between EGFR ligands and human chorionic gonadotropin (hCG) or human placental lactogen in amniotic fluid by ELISA. In addition, we also examined the EGFR ligands in syncytiotrophoblasts and the amount of hCG secretion in JAR, JEG3 and BeWo cells in the presence of each EGFR ligand.Key findingsIn order to identify possible candidates among the EGFR ligands, we examined the predominant expression of an EGFR ligand in the chorionic villi and amniotic fluid during normal pregnancy, and analyzed the relationship between EGFR ligands and hCG in trophoblastic model cells. Amphiregulin was primarily expressed throughout human pregnancy and stimulated the secretion of hCG, indicating that amphiregulin is a key molecule among EGFR ligands.SignificanceAmphiregulin may play a pivotal role in the development or maturation of placenta.     

Partial characterization of human choriocarcinoma cell line JAR cells in regard to oxidative stress

Characterization of free radical-induced cell injury processes of placenta cells is of vital importance for clinical medicine for the maintenance of intrauterine fetal life. The present study has analyzed cell injury processes in cells of the choriocarcinoma cell line JAR treated with menadione, an anticancer drug, and H(2)O(2) in comparison to osteosarcoma 143B cells using electron microscopic and flow cytometric techniques. Flow cytometry on JAR cells exposed to 100 muM menadione and double-stained with Annexin V and propidium iodide (PI) detected apoptotic cells reaching the maximum after 4 h of incubation with a rapid decrease thereafter. Viable cells became decreased to 46% of the control after 2 h of incubation, reaching 5% after 4 h. Cells stainable with both Annexin V and PI began to increase distinctly after 2 h of incubation, reaching 55% after 4 h. Electron microscopy showed that cells stainable with both dyes specified above had condensed nuclei and swollen cytoplasm, suggesting that they were undergoing a switch of the cell death mode from apoptosis to necrosis. On the other hand, 90% of 143B cells remained intact after 4 h of menadione treatment although the intracellular levels of superoxide were always higher than those of JAR cells treated with the drug. In contrast, JAR cells were more resistant than 143B cells to H(2)O(2)-induced cytotoxicity. These results may suggest that cytotoxicity of menadione cannot be explained simply by oxygen free radicals generated from the drug. The resistance of JAR cells to oxygen free radical-induced cytotoxicity may be advantageous for intrauterine fetal life.     

Effect of all-trans-retinoic acid on the differentiation, maturation and functions of dendritic cells derived from cord blood monocytes

We investigated the effects of all-trans-retinoic acid on dendritic cells derived from human cord blood monocytes to clarify how vitamin A affects immune function in children. Monocytes were separated from 18 cord blood samples, and dendritic cells were differentiated by culture. The percentage of dendritic cells was markedly lower in all-trans-retinoic acid treated cells than in untreated cells. After exposure to tumour necrosis factor-alpha for 3 days, all-trans-retinoic acid treated dendritic cells showed a reduced capacity to activate alloreactive T cells compared to untreated cells. In addition, all-trans-retinoic acid-treated dendritic cells could drive T cells towards T-helper cell type 2 responses with decreased secretion of interleukin-12, interferon-gamma, and increased production of interleukin-10 and interleukin-4. However, when Ro 41-5253, a selective retinoic acid receptor alpha antagonist, was add to culture, all the above actions were reversed. Thus, all-trans-retinoic acid may act at the first step of the immune response by inhibiting the differentiation of dendritic cells, maturation and induction of the T-helper cell type-2 response. The actions of all-trans-retinoic acid on dendritic cells were mediated through retinoic acid receptor alpha.     

Regulation of ADRP expression by long-chain polyunsaturated fatty acids in BeWo cells, a human placental choriocarcinoma cell line

Transplacental transfer of maternal fatty acids is critical for fetal growth and development. In the placenta, a preferential uptake of fatty acids toward long-chain polyunsaturated fatty acids (LCPUFAs) has been demonstrated. Adipose differentiation-related protein (ADRP) is a lipid droplet-associated protein that has been ascribed a role in cellular fatty acid uptake and storage. However, its role in placenta is not known. We demonstrate that ADRP mRNA and protein are regulated by fatty acids in a human placental choriocarcinoma cell line (BeWo) and in primary human trophoblasts. LCPUFAs of the n-3 and n-6 series [arachidonic acid (20:4n-6), docosahexaenoic acid (22:6n-3), and eicosapentaenoic acid (20:5n-3)] were more efficient than shorter fatty acids at stimulating ADRP mRNA expression. The fatty acid-mediated increase in ADRP mRNA expression was not related to the differentiation state of the cells. Synthetic peroxisome proliferator-activated receptor and retinoic X receptor agonists increased ADRP mRNA level but had no effect on ADRP protein level in undifferentiated BeWo cells. Furthermore, we show that incubation of BeWo cells with LCPUFAs, but not synthetic agonists, increased the cellular content of radiolabeled oleic acid, coinciding with the increase in ADRP mRNA and protein level. These studies provide new information on the regulation of ADRP in placental trophoblasts and suggest that LCPUFA-dependent regulation of ADRP could be involved in the metabolism of lipids in the placenta.     

hCG对绒癌细胞系MMP-2和MMP-8表达的影响

为了探讨人绒毛膜促性腺激素（hCG)对绒并且吕细胞侵袭性相关基因表达的影响作用。采用逆转录多聚酶链反应（RT-PCR）检测方法,观察了不同浓度hCG不同处理时间对JEG-3绒癌细胞系表达基质金属蛋白酶（MMP-2和MMP-8）的影响。结果显示,绒癌细胞系JEG-3表达MMP-2和MMP-8;分别用0、50、500、5000、25000IU/LhCG处理48h后,JEG-3细胞中MMP-2mRNA的含量无明显变化。MMP-8mRNA的表达则被诱导,并随hCG作用浓度增高而增强,进一步研究处理时间对MMP表达的影响。结果发现经25000IU/LhCG处理的JEG-3细胞,MMP-8的表达随处理时间的延长逐渐增强,而MMP-2的表达则在第6h被显著诱导后逐渐降低,以上结果提示,hCG可诱导绒癌细胞系JEG-3中MMP-2和MMP-8两种基质金属蛋白酶的表达,并因此可能对绒癌细胞系的侵袭性具有影响作用。然而hCG对这两者表达的影响规律并不完全一致。     

Effect of cyclic 3':5'-AMP derivatives prostaglandins and related agents on human chorionic gonadotropin secretion in human malignant trophoblast in culture.

The secretion of human chorionic gonadotropin (hCG) is stimulated by addition of N6, O2'-dibutyryl cyclic 3':5'-AMP (dbcAMP) or theophylline to normal term placenta and human malignant trophoblast cells in vitro. To understand better the specificity of this process, malignant trophoblast cultures were incubated with 3':5'-cyclic AMP (cAMP) derivatives, prostaglandins and other agents for 1 to 3 days, and the secretion of radioimmunoassayable hCG was measured. Whereas dbcAMP was the most potent agent in stimulating secretion of hCG, the N6--and O2'-monobutyryl derivatives of cAMP and phosphodiesterase inhibitors (theophylline, papaverine, 3-isobutyl-1-methylxanthine) also increased the secretion of the hormone. A slight increase in hCG secretion was observed following addition of adenine. By contrast, butyrate, cAMP, cyclic 3':5'-GMP (cGMP), dbcGMP, 5'-AMP, adenosine, L-epinephrine and prostaglandins E1, E2, F1a and F2a were ineffective. Particulate fractions from sonicates of malignant trophoblast cultures contained adenylate cyclase activity which was stimulated more than 10-fold by NaF, but not by either catecholamines or prostaglandins. The relatively specific stimulation of hCG secretion suggested that a regulatory process involving cAMP may have physiological significance in the trophoblast.