Liver X receptors mediate inhibition of hCG secretion in a human placental trophoblast cell line

Liver X receptors (LXR) alpha and beta are important regulators of lipid homeostasis in liver, adipose and other tissues. However, no such information is available for the human placenta. We determined expression of both LXR alpha and beta in placental trophoblast cell lines, BeWo and JAR. Exposure of BeWo cells to a synthetic LXR agonist, T0901317, resulted in an increase in the amount of mRNA of LXR target genes, sterol regulatory element-binding protein-1 and fatty acid synthase. T0901317 also increased the synthesis of lipids. Moreover, T0901317 resulted in a reduced secretion of hCG during differentiation of these cells. Our data for the first time demonstrate a new role for LXRs in the human placenta.     

Studies on influence of synthetic LH-RH and dbcAMP on normal chorionic villi and BeWo cells (author's transl)

The influence of synthetic LH-RH and dbcAMP on human chorionic villi and BeWo cells was studied through measurement of cAMP, hCG and estradiol. The results obtained were as follow: 1) There was a rapid increase of cAMP in chorionic villi after the 7th week of gestation, marking a maximum value at the 9th week, and gradually decreasing thereafter. 2) When dbcAMP was added to chorionic villi, there was a significant increase estradiol production in chorionic villi and hCG secretion in media. 3) The stimulation of cAMP in chorionic villi and hCG secretion into medial resulting from the addition of synthetic LH-RH to chorionic villi showed significant increases at 5 minutes and 30 minutes, respectively. 4) When dbcAMP or synthetic LH-RH was added to BeWo cells, increased secretion of hCG and estradiol into media was seen. It therefore is concluded that there is a relationship between the activity of synthetic LH-RH in which cAMP participates and a mechanism involving hCG secretion and estradiol production originating from human chorionic villi and BeWo cells. This is turn suggest the possibility of the existence in chorionic villi of a LH-RH like substance which plays a role in the production or secretion of hormone in the placenta.     

Long-term forskolin stimulation induces AMPK activation and thereby enhances tight junction formation in human placental trophoblast BeWo cells

BeWo cells, derived from human choriocarcinoma, have been known to respond to forskolin or cAMP analogues by differentiating into multinucleated cells- like syncytiotrophoblasts on the surfaces of chorionic villi of the human placenta. In this study, we demonstrated that long-term treatment with forskolin enhances the tight junction (TJ) formation in human placental BeWo cells. Interestingly, AMPK activation and phosphorylation of acetyl-CoA carboxylase (ACC), a molecule downstream from AMPK, were induced by long-term incubation (>12 h) with forskolin, despite not being induced by acute stimulation with forskolin. In addition, co-incubation with an AMPK inhibitor, compound C, as well as overexpression of an AMPK dominant negative mutant inhibited forskolin-induced TJ formation. Thus, although the molecular mechanism underlying AMPK activation via the forskolin stimulation is unclear, the TJ formation induced by forskolin is likely to be mediated by the AMPK pathway. Taking into consideration that TJs are present in the normal human placenta, this mechanism may be important for forming the placental barrier system between the fetal and maternal circulations.     

Differential effects of dibutyryl cyclic AMP and epidermal growth factor on the synthesis and secretion of human chorionic gonadotropin and its subunits by trophoblastic and non-trophoblastic cells

The effects of dibutyryl cyclic adenosine 3',5'-monophosphate (cAMP) and epidermal growth factor (EGF) on the synthesis and secretion of human chorionic gonadotropin (hCG) and its subunits by normal and malignant trophoblasts as well as by non-trophoblastic cells were investigated in vitro. The explants of normal early placental tissues, choriocarcinoma cell line BeWo and non-trophoblastic tumor cell line CaSki from epidermoid carcinoma of the cervix, respectively, were cultured in the presence or absence of dibutyryl cAMP or EGF. The addition of either dibutyryl cAMP (1 mM) or EGF (100 ng/ml) caused significant increases in the synthesis and secretion of hCG and its subunits in cultures of normal and malignant trophoblasts, but had no stimulatory effect on hCG beta synthesis and secretion in culture of non-trophoblastic cell line CaSki that secretes predominantly hCG beta-like material. The magnitude of the stimulatory effects of dibutyryl cAMP and EGF on hCG (alpha,beta) synthesis and secretion by BeWo cells was much greater than that observed in normal trophoblasts. The time course of these stimulatory effects indicated that EGF-stimulated increase in hCG synthesis and secretion required a lag period longer than that for the dibutyryl cAMP-stimulated increase. These results suggest that there were no differences in normal and malignant trophoblasts in the mechanism for the stimulatory regulation of hCG (alpha, beta) synthesis and secretion, but immunoreactive hCG beta synthesis and secretion in non-trophoblastic tumor cells are regulated by a mechanism different from that in trophoblastic cells.     

Regulation of Protein Expression and Function of OCTN2 in Forskolin-Induced Syncytialization in BeWo Cells

Placental OCTN2 is a high-affinity carnitine transporter that can interact with a number of therapeutic agents. The process of syncytialization is associated with the expression of a variety of genes. However, the association between syncytialization and OCTN2 expression is not yet clear. Given that forskolin induces BeWo cells to undergo biochemical and morphological differentiation, the purpose of the present study was to investigate whether the function and expression of OCTN2 are influenced by forskolin treatment during syncytialization.The forskolin-induced differentiation of BeWo cells was validated by secretion of β-human chorionic gonadotropin (β-hCG) and syncytin expression. Cellular localization of OCTN2 was analyzed by confocal microscopy. Expression of OCTN2 and the modular proteins PDZK1, PDZK2, NHERF1 and NHERF2 was analyzed by Western blotting and carnitine uptake by BeWo cells was estimated and the kinetic properties of uptake measured. The results showed that forskolin treatment increased β-hCG secretion and syncytin expression, suggesting induction of syncytialization. Confocal images of BeWo cells showed the localization of OCTN2 in the brush-border membrane. OCTN2 protein expression was upregulated in isolated brush-border membranes by long-term forskolin treatment, but the V_m for carnitine uptake was unchanged, although the K_m increased. PDZK1, NHERF1 and NHERF2 protein expression in the brush-border membrane was downregulated by forskolin treatment, whereas PDZK2 levels remained unchanged.In conclusion, protein expression and function of OCTN2 in BeWo cells can be regulated by forskolin treatment. While the presence of forskolin results in an increase in OCTN2 protein expression, the increase in uptake capacity may be compensated by the decreased expression of PDZK1, NHERF1 or NHERF2.     

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.     

Activation of adenosine A2B receptor impairs properties of trophoblast cells and involves mitogen-activated protein (MAP) kinase signaling

Introduction

Shallow trophoblast invasion of the maternal spiral arteries contributes to impaired placental perfusion and is hypothesized to be involved in the pathophysiology of preeclampsia. Hypoxia is a potent stimulus for the release of adenosine.

Methods

We investigated the effects of hypoxia and A_2B adenosine receptor signaling on migration, invasion, proteolytic activity of matrix metalloproteinase (MMP)-2, expression of MMP-2 and vascular endothelial growth factor (VEGF) mRNA, and production of human chorionic gonadotropin (hCG) in trophoblast cells (HTR-8/SVneo, BeWo).

Results

The adenosine A_2B receptor agonist 5-N-ethylcarboxamidoadenosine (NECA) reduced trophoblast (HTR-8/SVneo and BeWo) migration at 2%, 8% and 21% O₂ compared to untreated control cells. A_2B adenosine receptor stimulation decreased phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and stress-activated protein kinase/Jun-amino-terminal kinase (SAPK/JNK) at all three O₂ concentrations. ProMMP-2 activity, MMP-2 mRNA levels and hCG levels were markedly decreased after A_2B adenosine receptor activation in trophoblast cells. Adenosine receptor A_2B stimulation decreased VEGF expression at 2% and 8% O₂ but led to increased levels at 21% O₂.

Conclusions

These data indicate A_2B receptor activation blunts trophoblast migration possibly as a result of reduced activation of the MAPK signaling pathway and lower proMMP-2 levels. These data suggest a role for adenosine receptor A_2B in placental development and possibly in the pathophysiology of preeclampsia.     

Cystic fibrosis transmembrane regulator (CFTR) in human trophoblast BeWo cells and its relation to cell migration

Introduction

ENaC and CFTR are coexpressed in epithelia and have positive or negative functional interactions. In addition, ENaC and CFTR promote migration in placental trophoblastic cells and human airway cells, respectively. Here we tested the idea if CFTR is functionally expressed in BeWo cells, a trophoblastic cell line, and if it is involved in their migratory behavior.

Methods

CFTR expression was studied in BeWo cells with RT-PCR, biotinylation and Western blot. Ion currents were analyzed with patch clamp, and cell migration with the wound healing method.

Results

The mature CFTR 160-kDa band was present, and its localization at the surface membrane was confirmed. Forskolin (20 μM), an adenylate cyclase activator, was used for channel activation, and subsequently CFTR_inh-172 (2 μM) for its inhibition. The conductances in the presence of CFTR_inh-172 plus forskolin (16.0 ± 0.7 pS/pF and 32.6 ± 1.5 pS/pF) were significantly lower than in presence of only forskolin (29.7 ± 0.9 and 47.0 ± 2.0 pS/pF). The conductance of CFTR_inh-172 inhibited currents was 14.9 ± 0.7 pS/pF with a linear I-V relationship illustrating the nonrectifying properties of the CFTR. Cell migration was measured and covered 11.2 ± 0.4, 24.0 ± 1.7 and 13.9 ± 1.0% of the wound when cells were cultivated under control, forskolin, and forskolin plus CFTR_inh-172, respectively. Proliferation was not changed by any of the treatments.

Conclusions

Our results shows that BeWo cells functionally express the CFTR which plays a role in the wound healing increasing the cell migration process.     

Long-chain Polyunsaturated Fatty Acids Stimulate Cellular Fatty Acid Uptake in Human Placental Choriocarcinoma (BeWo) Cells

Supplementation of long-chain polyunsaturated fatty acids (LCPUFAs) is advocated during pregnancy in some countries although very little information is available on their effects on placental ability to take up these fatty acids for fetal supply to which the fetal growth and development are critically dependent. To identify the roles of LCPUFAs on placental fatty acid transport function, we examined the effects of LCPUFAs on the uptake of fatty acids and expression of fatty acid transport/metabolic genes using placental trophoblast cells (BeWo). Following 24 h incubation of these cells with 100 μM of LCPUFAs (arachidonic acid, 20:4n-6, eicosapentaenoic acid, 20:5n-3, or docosahexaenoic acid, 22:6n-3), the cellular uptake of [¹⁴C] fatty acids was increased by 20–50%, and accumulated fatty acids were preferentially incorporated into phospholipid fractions. Oleic acid (OA, 18:1n-9), on the other hand, could not stimulate fatty acid uptake. LCPUFAs and OA increased the gene expression of ADRP whilst decreased the expression of ASCL3, ACSL4, ACSL6, LPIN1, and FABP3 in these cells. However, LCPUFAs but not OA increased expression of ACSL1 and ACSL5. Since acyl-CoA synthetases are involved in cellular uptake of fatty acids via activation for their channelling to lipid metabolism and/or for storage, the increased expression of ACSL1 and ACLS5 by LCPUFAs may be responsible for the increased fatty acid uptake. These findings demonstrate that LCPUFA may function as an important regulator of general fatty acid uptake in trophoblast cells and may thus have impact on fetal growth and development.     

Human placental gonadotrophin-releasing hormone (GnRH) binding sites: I. Characterization, properties and ligand specificity

Radioiodinated gonadotrophin-releasing hormone tracers were prepared from mammalian (m GnRH), salmon (s GnRH), lamprey (l GnRH) and the two forms of chicken GnRH (ch GnRH I and ch GnRH II), and also from the GnRH agonist (GnRHA) analogues, Buserelin ([D-Ser(tBu)6] 1-9 GnRH ethylamide) and Tryptorelin ([D-Trp6 GnRH] 1-9 ethylamide) and a GnRH antagonist (GnRHANT; [Ac 3,4-dehydro-Pro1, D-p-F-Phe2, D-Trp3,6] LHRH). Specific binding of hormone tracers was compared in homogenates and membrane fractions from human placenta and rat pituitary. GnRH agonist tracers bound readily to pituitary and placental binding sites. Binding of m GnRH to rat pituitary membranes was low compared to agonist binding, whereas other GnRH iso-forms were not bound. Binding of 125I-labelled m GnRH to human placental membranes was also low compared to that of Buserelin, and l GnRH and ch GnRH I tracers bound poorly. However, human placental membranes bound s GnRH and ch GnRH II to the same extent as GnRHA. Studies of the inactivation of GnRH tracers following incubation with rat pituitary and human placental membranes demonstrated that, although GnRH isoforms were degraded at different rates in these tissues, the differential ability of GnRH isoforms to bind to pituitary or placental binding sites was not related to differences in degradation of tracers, but rather to differences in ligand specificity. Specific binding of 125I-labelled GnRH agonists (GnRHA) and mammalian GnRH (m GnRH), s GnRH and ch GnRH II tracers to human placental membrane fractions increased linearly with increasing membrane protein at low concentrations. Binding was dependent on both the duration and temperature of incubation, and pH profiles of 125I-labelled GnRHA, s GnRh and ch GnRH II binding to placental membranes were similar. Once bound s GnRH formed a tighter complex with placental receptors than GnRHA, though 125I-labelled s GnRH was inactivated more rapidly than agonist tracer during incubation with placental membranes. Binding of GnRH tracers was specific for molecules with the GnRH structure. Deletions of amino acid residues at positions 1-3 and/or deamidation at Gly10 reduced binding potencies for both human placental and rat pituitary binding sites, indicating that both ends of the GnRH molecule were required for optimal binding. Modifications which conferred increased agonist activity led to markedly increased receptor binding potency in the rat pituitary, but only slightly increased potency for placental receptors. In contrast, GnRH antagonist analogues had increased potency towards pituitary receptors, but much reduced potency towards human placental binding sites.(ABSTRACT TRUNCATED AT 400 WORDS)     

Hormonal regulation of galectin 3 in trophoblasts and its effects on endometrium

Previous studies had shown important functions of galectin 3 (Gal-3) in endometrium during embryo implantation, in regulation of endometrial cell proliferation and adhesion by interacting with integrin β3. In this study, we investigated hormonal regulation of Gal-3 in trophoblasts and its extracellular effects on endometrium. We used BeWo and RL95-2 cells as a model of trophoblastic and endometrial epithelial cells, respectively, to create an in vivo model of embryo implantation. Our results indicated that 17β-estradiol (E2), progesterone (P4), and human chorionic gonadotropin (hCG) induced the expression of Gal-3 and promoted its secretion from BeWo cells. The exogenous Gal-3 inhibited cell proliferation and induced apoptosis of endometrial cells (RL95-2 cells) through activation of integrin β1. We further validated the proapoptotic effect of Gal-3 secreted by trophoblastic cell on endometrial cells by culturing RL95-2 cells with Bewo cells and measuring the apoptotic rate. Our analysis provides new insight into the critical roles of Gal-3 in embryo implantation.     

Transplacental transfer of melamine

Objective

To characterize transplacental transfer of melamine and related mechanisms as well as toxicity using human placental perfusion and cultured cells.

Methods

Transfer and toxicity were analyzed in 4-h perfusions with 10 μM or 1 mM melamine, or 10 μM melamine with 10 nM cyanuric acid (CYA). Efflux transporters were studied in accumulation assay and toxicity in BeWo cells by MTT assay.

Results

Of added melamine 34-45% was transferred to fetal circulation and CYA made no difference. Histology, hCG production, and PLAP activity indicated functionality of placental tissue with no grave toxicity. Highest concentration of melamine used (2 mM) with CYA and long treatment time decreased viability of BeWo cells. Inhibitors of ABCB1, ABCG2, ABCC2 did not affect the accumulation of melamine in cells.

Conclusion

Melamine goes through human term placenta with no contribution of efflux transporters. Toxicity of melamine is low in placental tissue and BeWo cells.     

Expression of NADPH oxidase isoform 1 (Nox1) in human placenta: involvement in preeclampsia

Increased oxidative stress in the placenta has been associated with preeclampsia (PE), a clinical syndrome involving placental pathology. The enzymatic sources of reactive oxygen species in the human placenta are as yet unidentified. We hypothesized that NADPH oxidase is a main source of reactive oxygen species in the placenta and its expression may change in PE. Employing RT-PCR, we have amplified a novel NADPH oxidase isoform Nox1 from human choriocarcinoma BeWo cells. Using polyclonal anti-peptide antiserum recognizing unique Nox1 peptide sequences, we identified by immunohistochemistry and cell fractionation that Nox1 protein localizes in the BeWo cell membrane structures. Immunohistochemistry of normal placental tissues showed that Nox1 was localized in syncytiotrophoblasts, in villous vascular endothelium, and in some stromal cells. At the immunohistochemical level Nox1 expression was significantly increased in syncytiotrophoblast and endothelial cells in placentas from patients with preeclampsia as compared to gestational age-matched controls. Western blot analysis of whole placental homogenate confirmed this increase. Our data suggests that increased Nox1 expression is associated with the increased oxidative stress found in these placentas.     

Effect of methotrexate on cell growth and the production of hCG, beta-hCG and SP-1 in cultured choriocarcinoma cell lines

Three choriocarcinoma cell lines (BeWo, GCH-2 and SCH) cultured in MTX or MTX-free medium were investigated with respect to cell growth and production of hCG, beta-hCG and specific beta-1-glycoprotein (SP-1). MTX was administered at the concentration of 10(-8), 10(-7), 10(-6), 10(-5)M for 24 hours, 72 hours and 168 hours from the fourth day of culture. Each cell line demonstrated that the cell growth and production of hCG and beta-hCG were dose, rather than time, dependent. 10(-6)M or more of MTX had a distinct effect, but 10(-7) or less of MTX had little effect. SP-1 was produced at a low level during culture regardless of the medium used. The ratio of the extra- to the intracellular hCG level was shown to be about 100:1 after the administration of MTX to BeWo and GCH-2. It is, therefore, possible that the temporary elevation of the hCG level observed in vitro after treatment with MTX is the result of an increase in secretion of hCG, rather than cellular damage.     

Macrophage-activating lipopeptide-2 induces cyclooxygenase-2 and prostaglandin E(2) via toll-like receptor 2 in human placental trophoblast cells

We have examined whether toll-like receptor (TLR)2-mediated stimulation by macrophage-activating lipopeptide-2 (MALP-2), originally purified from Mycoplasma fermentans, induces cyclooxygenase (COX)-2 and prostaglandin (PG)E(2) in human placental trophoblast cells. The signaling mechanism by which MALP-2 exerts its effect has also been examined. Human placental trophoblast cells isolated from term placenta were used. TLR expression in trophoblast cells was confirmed by multiplex PCR and immunocytochemistry, and examined whether MALP-2 induces COX-2 and PGE(2) by Northern blotting, RT-PCR, Western blotting and ELISA, respectively. The activation of NF-kappaB and MAP kinases (ERK1/2 and p38) was examined by Western blotting. The effects of inhibitors of NF-kappaB, MEK1/2 and p38 on MALP-2-induced PGE(2) production were also evaluated. TLR2, TLR6 and TLR4 were expressed in human placental trophoblast cells. MALP-2 significantly induced COX-2 expression and enhanced PGE(2) production in a dose-dependent manner. MALP-2 induced the activation of NF-kappaB, ERK1/2 and p38 MAPK. Inhibitors of NF-kappaB, MEK1/2 and p38 blocked MALP-2-inducible PGE(2) production. TLR2-mediated stimulation by MALP-2 induces COX-2 and PGE(2) in human placental trophoblast cells via NF-kappaB and MAP kinases pathways.     

Human placental gonadotrophin-releasing hormone (GnRH) binding sites: II. Comparison of binding and inactivation of I-labelled GnRH agonist to subcellular fractions following density gradient centrifugation

Human placental homogenates and membrane fractions were centrifuged on continuous sucrose density gradients, with or without buoyant density perturbation of plasma-membranes by digitonin, and aliquots of each gradient fraction were assayed for a range of plasma-membrane and intracellular organelle markers, and for specific binding and inactivation of radiolabelled GnRH agonist (GnRHA), Buserelin ([D-Ser(tBu)6] GnRH 1-9 ethylamide). GnRH agonist (GnRHA) binding equilibrated in the same regions of control gradients as the plasma-membrane markers, EGF-receptor and alkaline phosphatase. Moreover, binding of 125I-labelled GnRHA and 125I-labelled chicken GnRH II (ch GnRH II) was enriched in the same regions of the gradients, indicating that both bound to the same membrane fractions. Digitonin pretreatment increased the buoyant density of all three placental plasma-membrane markers to a similar degree. Intracellular organelle markers (and hCG content) equilibrated in different regions of the gradient to placental surface-membranes, and were not perturbed appreciably by digitonin. In contrast, inactivation of 125I-labelled GnRHA was associated largely with the soluble (cytosol) fraction which failed to enter the gradient, and little tracer inactivation was observed in fractions enriched in GnRHA binding activity. Similar results were obtained with fractionated rat pituitary membranes. We conclude that: (a) placental GnRH binding sites do not represent binding of radiolabelled ligand to GnRH-degrading enzymes, (b) degradation of radiolabelled ligand is associated largely with placental cytosol fractions, and (c) GnRH binding activity appears to be associated largely with placental plasma-membranes.