Upregulation of neutral endopeptidase expression and enzymatic activity during the differentiation of human choriocarcinoma cells.

Neutral endopeptidase (NEP)/CD10, a cell-surface peptidase degrading various bioactive peptides, is mainly present in syncytiotrophoblasts in the human placenta. However, the change in NEP expression upon trophoblast differentiation remains to be clarified. In the present study, we examined the expression of NEP in the differentiating trophoblast using the BeWo choriocarcinoma cell line as a model system. Under the normal culture conditions, NEP was very weakly expressed on most proliferating cytotrophoblastic BeWo cells, while a minority of the cell population (less than 5 per cent ), consisting of giant, multinucleated cells, clearly expressed NEP at the cell membrane. Treatment of BeWo cells with forskolin (FSK) for 48-72 h resulted in an 11- to 44-fold increase in the level of hCG secretion and induced cell fusion leading to the formation of multinucleated syncytiotrophoblasts, indicating functional and morphological differentiation. Fluorescence-activated cell sorting (FACS) analysis revealed that treatment with FSK significantly increased the cell-surface protein expression of NEP on differentiating BeWo cells. Consistently, there was a significant increase in the NEP enzymatic activity after FSK treatment. The level of hCG secretion from the FSK-treated cells was further enhanced when the cells were treated in the presence of the NEP inhibitor phosphoramidon. Immunohistochemical analysis of normal chorionic villi and choriocarcinoma tissues revealed the localization of NEP in syncytiotrophoblastic cells, as opposed to weak or negative staining in cytotrophoblastic cells. These data demonstrate that induction of choriocarcinoma cell differentiation is associated with an increase of NEP/CD10 expression at the cell surface, suggesting a role of this enzyme in regulating differentiated trophoblast functions such as hCG secretion. NEP/CD10 may also be a new cellular differentiation marker of both the normal and neoplastic trophoblast.     

Signal pathway involved in increased expression of neutral endopeptidase 24.11 by gonadotropin releasing hormone in choriocarcinoma cells

Neutral endopeptidase 24.11 (NEP) is known to regulate cellular functions by degrading several bioactive peptides, such as gonadotropin-releasing hormone (GnRH). The present study was performed to clarify the mechanisms of NEP expression by GnRH in human choriocarcinoma (BeWo) cells. GnRH increased NEP expression and enzyme activity in a dose- and time-dependent manner in BeWo cells. The phosphorylation levels of protein kinase C (PKC) delta, p38 mitogen-activated protein kinase (MAPK), and c-Jun N-terminal kinase (JNK1 and 2) were enhanced after 10 min exposure of 10(-6)m GnRH. The effect of GnRH on both NEP expression and enzyme activity was completely inhibited by inhibitors of PKC, PKC delta, and p38MAPK. Cell number was reduced by 54.4 per cent of the control by culture with 10(-6)m GnRH for 24 h. However, phosphoramidon, a NEP specific inhibitor, inhibited antiproliferative effect of GnRH and reverted to the control level. In conclusion, GnRH induces NEP expression by PKC delta and p38MAPK, and increased NEP expression may be involved in antiproliferative effect in BeWo cells.     

The cellular mechanism by which the human endogenous retrovirus ERV-3 env gene affects proliferation and differentiation in a human placental trophoblast model, BeWo

The env region of the human endogenous retrovirus ERV-3 is expressed during differentiation of trophoblast and the choriocarcinoma BeWo. Stable transfectants with ERV-3 env exhibit most aspects of trophoblast differentiation, including inhibition of cell proliferation, changes in cell morphology, and increased production of beta-hCG mRNA. In this study, the cellular mechanism of induction of BeWo cell differentiation by ERV-3 env was investigated. In BeWo cells stably transfected with ERV-3 env, the production of beta-hCG mRNA and hCG protein was increased. Intracellular cAMP level was markedly increased over that of vector transfected cells. The effect on beta-hCG protein production was inhibited by H89, a protein kinase A (PKA) inhibitor, while protein kinase C (PKC) and protein tyrosine kinase (PTK) inhibitors had no effect. The expression of a major cell cycle promoter, cyclin B, was markedly reduced while expression of p21, a negative regulator of the cell cycle, was up-regulated. Inhibition of ERV-3 env induced hCG production with H89 had no significant effect on cell growth when compared with cells transfected with vector alone.     

STAT3-mediated constitutive expression of SOCS3 in an undifferentiated rat trophoblast-like cell line

In the trophoblast, constitutive expression of SOCS3 is important for the negative regulation of trophoblast giant cell differentiation. In this study, we analyzed the signaling pathway regulating the constitutive SOCS3 expression in undifferentiated Rcho-1 cells, which were derived from rat choriocarcinoma and consist of trophoblast stem cells that are capable of differentiating to trophoblast giant cells in vitro. PD98059, an MEK inhibitor, repressed the SOCS3 expression but AG490, a JAK2 inhibitor, did not. Promoter deletion analysis revealed that the STAT response element (SRE) in the SOCS3 promoter is necessary for the promoter activity. Overexpression of STAT3 increased the SOCS3 promoter activity, whereas expression of dominant-negative STAT3 reduced it. Constitutive STAT3 tyrosine phosphorylation that was not inhibited by either AG490 or PD98059 was demonstrated. Electrophoretic mobility shift assays showed the existence of a protein that bound to SRE and was supershifted with STAT3 antibody. This binding reaction was inhibited by neither AG490 nor PD98059. These findings imply that the ERK/MAPK pathway and STAT3 are involved in the constitutive activation of SOCS3 in undifferentiated Rcho-1 cells. Moreover, they indicate that the constitutive STAT3 tyrosine phosphorylation and the DNA binding activity of STAT3 do not depend on the ERK/MAPK or JAK kinase pathway. These results suggest that a trophoblast-specific STAT3 activation pathway is important for the regulation of giant cell differentiation.     

Caspase-14: a new player in cytotrophoblast differentiation

The human placenta is responsible for the exchange of nutrients, gas and wastes through the trophoblast maternal-fetal barrier, which is formed by the fusion of villous cytotrophoblasts to form the continuous multinucleated syncytiotrophoblast separating the maternal and fetal circulations. Caspase-14 is a seemingly non-apoptotic caspase involved in keratinocyte differentiation and cornification. It is proposed that caspase-14 has a conserved role in cellular differentiation and a role in differentiation and fusion in the trophoblast. The human choriocarcinoma BeWo cell line was treated with staurosporine and forskolin to induce apoptosis and differentiation respectively. Staurosporine initiated apoptosis within 3 h of treatment, while apoptosis was completed following 6 h treatment. Caspase-14 gene and protein expression was unchanged throughout this process. During BeWo differentiation, caspase-14 mRNA was elevated after 48 h forskolin treatment, while its protein was increased after 24 h. Therefore, caspase-14 is up-regulated during trophoblast differentiation, as represented by the BeWo cell line. Moreover, caspase-14 may interact with other signalling molecules to facilitate differentiation. This new data confirms the potential for the BeWo cell line in the functional dissection of this unusual caspase and its prospective role in trophoblast differentiation.     

Molecular regulation of human placental growth factor (PlGF) gene expression in placental villi and trophoblast cells is mediated via the protein kinase a pathway

Cyclic 3',5'-adenosine monophosphate (cAMP) is a critical second messenger for human trophoblasts and regulates the expression of numerous genes. It is known to stimulate in vitro the fusion and differentiation of BeWo choriocarcinoma cells, which acquire characteristics of syncytiotrophoblasts. A DNA microarray analysis of BeWo cells undergoing forskolin-induced syncytialization revealed that among the induced genes, placental growth factor (PlGF) was 10-fold upregulated. We verified this result in two choriocarcinoma cell lines, BeWo and JEG-3, and also in first trimester placental villous explants by quantifying PlGF mRNA (real time PCR) and PlGF protein secreted into the supernatant (ELISA). Similar effects were noted for vascular endothelial growth factor (VEGF) mRNA and protein expression. Treatment with cholera toxin and the use of a specific inhibitor of protein kinase A (PKA) blocked these effects, indicating that the cAMP/PKA pathway is responsible for the cAMP-induced upregulation of PlGF and that one or more G protein coupled receptor(s) was involved. We identified two functional cAMP responsive elements (CRE) in the PlGF promoter and demonstrated that the CRE binding protein, CREB, contributes to the regulation of PlGF gene expression. We speculate that defects in this signaling pathway may lead to abnormal secretion of PlGF protein as observed in the pregnancy-related diseases preeclampsia and intrauterine growth restriction.     

Phosphorylation of JAK2 by serotonin 5-HT (2A) receptor activates both STAT3 and ERK1/2 pathways and increases growth of JEG-3 human placental choriocarcinoma cell

Serotonin 5-HT_2A receptor activation improves viability, increases DNA synthesis and activates JAK2-STAT3 and MEK1/2-ERK1/2 signalling pathways in JEG-3 human trophoblast choriocarcinoma cells. The goal of this study was to characterize the signal transduction cascade involved in 5-HT_2A receptor-induced growth of JEG-3 cells. Selective 5-HT_2A receptor agonist, DOI, induced JEG-3 cell growth was inhibited by the inhibitor of JAK2 (AG490), MEK1/2 (U0126), phospholipase C-β (PLC-β; U73122) and protein kinase C-β (PKC-β; Gö6976)), whereas the selective phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002) had no effect. Specific inhibitors of PLC-β, PKC-β and Ras (farnesylthiosalicylic acid) inhibit activation of ERK1/2, whereas the PKC-ζ inhibitor GF109203X had no effect. Interestingly, inhibition of JAK2 prevented DOI-induced phosphorylation of ERK1/2 whereas inhibition of ERK1/2 pathway had no effect on DOI-induced activation of STAT3. Taken together, our results demonstrate that both the JAK2-STAT3 and PLC-β-PKC-β-Ras-ERK1/2 signalling pathways are involved in the stimulation of JEG-3 cell growth mediated by DOI. Moreover, this study shows that activation of JAK2 by the 5-HT_2A receptor is essential to activate both STAT3 and ERK1/2 signalling pathways as well as to increase JEG-3 choriocarcinoma cell growth and survival.     

Calcitonin promotes outgrowth of trophoblast cells on endometrial epithelial cells: involvement of calcium mobilization and protein kinase C activation

Embryo implantation is a complex process that requires coordinated trophoblast-endometrial interactions. During implantation, trophoblast cells of the attached blastocyst penetrate the luminal epithelium of the endometrium before invasion into the endometrial stroma. Previous studies demonstrated that calcitonin was actively secreted by rat and human endometrial epithelial cells (EEC) during the implantation window and targeted disruption of endometrial calcitonin expression dramatically decreased embryo implantation rates; however, the role and signal transduction of calcitonin in trophoblast-endometrial interactions remained unclear and are therefore examined in this study. BeWo trophoblast and RL95-2 EEC lines were used because they preserve many properties of their respective normal tissues. We co-cultured BeWo trophoblast spheroids with RL95-2 EEC monolayers to mimic the blastocyst-endometrial interaction, and found that most spheroids quickly attached to EEC monolayers and then progressively expanded, with marked displacement of EEC adjacent to the outgrowing trophoblast cells. Interestingly, pretreatment of EEC monolayers with calcitonin before the addition of spheroids significantly enhanced trophoblast expansion on EEC monolayers. Cytosolic calcium (Ca(2+)) levels in EEC increased rapidly upon exposure to calcitonin, and blockade of Ca(2+) release by BAPTA-AM effectively prevented the promoting effect of calcitonin on trophoblast expansion on EEC. The Ca(2+)-dependent protein kinase C (PKC) was also activated in EEC after calcitonin treatment, and the PKC inhibitors staurosporine and calphostin C could completely abolish calcitonin-induced augmentation of trophoblast expansion on EEC. Our results suggest that calcitonin promotes trophoblastic displacement of EEC through calcium mobilization and PKC activation, thereby facilitating embryo implantation.     

Cytotoxic effect of zinc-citrate compound on choriocarcinoma cell lines

This study investigated the cytotoxic effect of zinc-citrate compound (CIZAR) on choriocarcinoma cell lines. Primary cultured normal trophoblast cells (NPT), human tumorigenic poorly differentiated trophoblast cell line (HT), and choriocarcinoma cell line (BeWo) were exposed to different concentrations of CIZAR and cultured at different times. Cell viability was determined by CCK-8 assay. The effects on cell cycle progression, population distribution and apoptotic incidence were determined by flow cytometry. The appearance of apoptosis was confirmed by DNA laddering and DAPI staining. The quantitative analysis of telomerase was measured by TRAPeze telomerase detection kit. The molecular mechanism of CIZAR-induced apoptosis was examined with Western blot analysis and colorimetric caspase-3 activity assay. In in vitro condition, CIZAR had a selective cytotoxic effect on choriocarcinoma cell line in dose- and time-dependent patterns. Flow cytometric analysis, DNA laddering, and DAPI staining indicated that BeWo cells only have been induced apoptosis by CIZAR. Shortening of telomere was also observed only in BeWo cells. Results also displayed that CIZAR-induced apoptosis involves the up-regulation of p21(WAF1) and Bax protein and down-regulation of Bcl-2 which were accompanied by the activation of caspase-3. Taken together, our results suggest that CIZAR is an apoptotic inducer in malignant trophoblast cells (BeWo).     

PP2 regulates human trophoblast cells differentiation by activating p38 and ERK1/2 and inhibiting FAK activation

Throughout gestation, fetal growth and development depend, in part, on placental transfer of nutrients from the maternal circulation. This latter function depends on multinucleated, terminally differentiated syncytiotrophoblasts. In vitro, freshly isolated cytotrophoblast cells differentiate spontaneously into syncytiotrophoblast in the presence of fetal bovine serum (FBS). We have previously showed that trophoblast differentiation is regulated by ERK1/2 and p38. Moreover, we showed that PP2 [4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3, 4-d]pyrimidine], a Src family kinase (SFK) specific inhibitor, stimulates biochemical trophoblast cells differentiation while it inhibits cell adhesion and spreading without affecting cell fusion. Therefore, we examined the mechanisms by which PP2 modulates trophoblast cells differentiation. This study shows that PP2 stimulates ERK1/2 and p38 activation after 24h of treatments and up to 3 days while it inhibits focal adhesion kinase (FAK) phosphorylation at many sites including Tyr-397, 407, 576 and 577. Furthermore, we showed that transient activation of ERK1/2 by FBS is independent of SFK and that PP2 induces rapid activation of p38. Moreover, the kinase activity of SFK is negatively regulated by the phosphorylation of their carboxy (C)-terminal regulatory tyrosines by specific proteins called carboxyl-terminal Src kinase (Csk) and Csk homologous kinase (CHK). We showed the expression of Csk and CHK in human trophoblast cells. In summary, this study showed that PP2 stimulates the biochemical differentiation of trophoblast cells by stimulating p38 and ERK1/2 while it inhibits the morphological differentiation by inhibiting FAK activation.     

Immunohistochemical studies of fetal trophoblast and maternal decidua in hydatidiform mole and choriocarcinoma

Immunohistochemical techniques have been used to investigate the expression of fetal trophoblast antigens and the maternal leucocytic response in molar pregnancy and choriocarcinoma. The antigenic phenotype of morphologically defined trophoblast populations in complete, partial and invasive moles was analogous to that in normal pregnancy. All trophoblast phenotypes described in normal pregnancy were also identified in choriocarcinoma, suggesting that extensive differentiation into heterogeneous subgroups occurs in malignant trophoblast. The maternal leucocytic infiltrate in molar pregnancy consisted of T lymphocytes and class II MHC-positive macrophages. CD2-positive, CD3-negative lymphocytes were identified in molar decidua but not in uterine tissue in choriocarcinoma. Similarly, endometrial granulocytes were present in molar decidua but not in choriocarcinoma; these cells were associated with decidualization rather than with fetal trophoblast.     

Glycodelin-A modulates syncytialization of human BeWo choriocarcinoma cell line

Cytotrophoblasts are the key trophoblast cells which differentiate into different trophoblast lineages. In this report, glycodelin-A action on fusion of a cytotrophoblast-like cell line (BeWo) was investigated. It significantly reduced the spontaneous fusion of BeWo cells. The treatment enhanced the invasion and extracellular-signal regulated kinases activation of BeWo cells. The mRNA expression of syncytialization markers, human chorionic gonadotrophin and glial cells missing homolog 1 were suppressed upon glycodelin-A treatment. The data suggest a possible function of glycodelin-A in mediating cytotrophoblast differentiation.     

The differentiation antigen of trophoblast expressed on trophoblastic neoplasms (choriocarcinoma and hydatidiform mole) detected by monoclonal antibodies

To analyse the differentiation and malignant transformation of human trophoblasts, attempts were made to establish monoclonal antibodies (MoAbs) that react with cells at a certain stage of the trophoblast differentiation process. Two MoAbs, TM7-3 and TM3-8, were obtained by immunizing BALB/c mice with a human choriocarcinoma cell line, BeWo. In the cellular radioimmunoassay with 125I-labelled F(ab')2 of goat anti-mouse IgG, they reacted with nine out of ten choriocarcinoma cell lines and five of sixteen non-choriocarcinoma cell lines but not with twenty-six lymphoblastoid cell lines tested. Immunofluorescence and immunoperoxidase stainings showed that these MoAbs reacted predominantly with cytotrophoblast-like, but not syncytiotrophoblast-like components in the tissue of choriocarcinoma or hydatidiform mole, in the normal chorionic villi, cytotrophoblasts were very weakly positive but syncytiotrophoblasts were negative, and in a variety of normal tissues tested, only a part of the urinary tubules of kidney and the ducts of pancreas reacted to these MoAbs. These results suggest that TM7-3 and TM3-8 detect a differentiation antigen of human trophoblasts, which appears to increase with the malignant transformation of cytotrophoblasts. Thus, TM7-3 and TM3-8 may be useful in the study of human trophoblast differentiation.