Syncytin-1 modulates placental trophoblast cell proliferation by promoting G1/S transition

Placental syncytiotrophoblasts formed by the fusion of cytotrophoblasts constitute the interface between maternal and fetal circulations. The syncytium, composed of a continuous layer of syncytiotrophoblasts, assumes the fetal–maternal nutrient exchange, placental barrier, and endocrine functions important for the maintenance of normal pregnancy. Syncytin-1, an endogenous retroviral gene product, mediates the fusion of cytotrophoblasts. While the fusogenic function of syncytin-1 has been well established, little is known regarding its nonfusogenic activities. This study investigates the role of syncytin-1 in trophoblast proliferation. We found that syncytin-1 knockdown significantly inhibited BeWo cell growth and DNA synthesis. Moreover, time course studies on key cell cycle regulators demonstrated an upregulation of p15 and downregulation of CDK4, E2F1, PCNA, and c-Myc, which consequently led to a reduced level of CDK1. These results, together with those from flow cytometry analysis, indicated that syncytin-1 knockdown blocked the G1/S transition phase of the cell cycle. Moreover, syncytin-1 overexpression promoted CHO cell proliferation and led to changes opposite to those observed in syncytin-1 knockdown experiments, confirming the critical role of syncytin-1 for G1/S transition. Thus, syncytin-1, through both nonfusogenic and fusogenic, functions, may co-regulate the input (proliferation) and output (fusion) of the cytotrophoblast “pool”. Such co-regulation could be an efficient way to achieve the balance between these two opposing processes, which is required for syncytium homeostasis. Since decreased syncytin-1 expression has been shown to be associated with preeclamptic and hypoxic condition, insufficient replenishing of the cytotrophoblast “pool” may contribute to syncytium deficiency, a critical pathological change frequently found in preeclamptic placentas.     

Angiotensin II mimics the hypoxic effect on regulating trophoblast proliferation and differentiation in human placental explant cultures

Regulation of cytotrophoblast differentiation toward extravillous trophoblasts (EVTs) is critical for establishing successful pregnancy. Previous studies have focused primarily on the factors promoting the differentiation, while inhibitory regulators except hypoxia have been less documented. In this study, to test our hypothesis that angiotensin II (Ang II) would inhibit EVT differentiation, we investigated the effects of Ang II on trophoblast outgrowth and the expression of molecules associated with the proliferation and invasion of trophoblasts using human first trimester villous explant cultures. Ang II increased EVT outgrowth and the number of cells in cell columns. Moreover, Ang II-treated explants exhibited increased Ki67 and integrin alpha5 immunoreactivity in EVTs as well as matrix metalloproteinase-2 activity in the conditioned media, and decreased alpha1 integrin immunoreactivity, which are compatible with the features of the proliferative phenotype EVTs. These effects of Ang II were similar to those of hypoxia (3% O(2)). Ang II stimulated the expression of hypoxia inducible factor-1alpha at both mRNA and protein levels, and also enhanced the expression of plasminogen activator inhibitor-1 (PAI-1). Data presented herein suggest a possible role for Ang II in impairing trophoblast differentiation toward an invasive phenotype, which might be associated with shallow invasion in preeclamptic placentas.     

Glucocorticoid binding to solubilized components of human placental trophoblast membranes

We have recently described glucocorticoid uptake by human placental membrane vesicles which is specific, saturable and has a low K (7 nM). This paper describes solubilization of these vesicles with Triton x-100 and successful demonstration of glucocorticoid binding to the putative transport site. This was accomplished by analysis of corticosterone binding to the 140,000 × g supernatant of solubilized vesicles using G-75 Sephadex chromatography. The amount of bound corticosterone present in the void volume was proportional to the concentration of solubilized vesicles. The specificity of binding was tested by coincubation of tritiated corticosterone with 100-fold excesses of various steroids. The relative ability of various steroids to inhibit binding was corticosterone=pregesterone- >aldosterone. Triamcinolone acetonide, and estradiol were ineffective competitors. We conclude from these studies that human placental membranes contain glucocorticoid-specific binding sites which can be solubilized with Triton x-100. It is possible that these sites are involved in membrane mediated transport of glucocorticoids by this tissue.     

Insulin-like growth factor-1 stimulates amino acid uptake by the cultured human placental trophoblast

Amino acid uptake by the human placenta is known to occur via several transport mechanisms. However, regulation by extracellular factors has received relatively little attention. A recent report by this laboratory characterized the uptake of α-aminoisobutyric acid (AIB) stimulated by insulin in the cultured human placental trophoblast The current study evaluated the effect of insulin-like growth factor-1 (IGF-1) on AIB uptake in cultured human placental trophoblasts. Na⁺-dependent AIB uptake was significantly stimulated by IGF-l in a time-dependent manner, as early as 30 min after hormone exposure. The maximum effect was at 2–4 hr of continuous exposure to IGF-l and the stimulation was dependent upon IGF-1 concentration approaching maximal stimulation at 50 ng.ml^?1. AIB uptake was inhibited by increasing concentrations of α-(methylamino)isobtyric acid (MeAIB). Approximately 75% of basal (unstimulated) Na⁺-dependent AIB uptake was inhibited by MeAIB. The IGF-1-stimulated increment above basal AIB uptake was completely inhibited by MeAIB. IGF-1 increased the maximum uptake yelocity but not Km. Using equimolar concentrations, stimulation was greater with IGF-1 then with IGF-2. Stimulation by IGF-1, but not insulin, was inhibited by anit-IGF-1 receptor antibody, indicating mediation via the IGF-1 receptor. H7, a nonspecific inhibitor of serine-threonine kinase, inhibited IGF-1-dependent stimulation of AIB uptake. In addition, calphostin C (a specific inhibitor of protein kinase C), but not H89 (a specific inhibitor of protein kinase A), inhibited the IGF-1 action. This study further characterizes regulated amino acid uptake by the human placental trophoblasts and demonstrates that the Na⁺-dependent component of AIB uptake is stimulated by physiologic concentrations of IGF-1. © 1995 Wiley-Liss Inc.     

Two pathways of placental lactogen secretion by cultured human trophoblast

To assess the relative importance of regulated and of constitutive secretion of placental lactogen, a cell culture model of term human trophoblast was utilized. Time courses of secretion revealed a constant secretion rate over 9 days of culture, with relatively small constant intracellular hormone concentration. Potassium, 21 mM, produced a slight but significant increase in hormone secretion into the medium. Growth hormone-releasing hormone (5 X 10(-10)-5 X 10(-9)) stimulated a 27-48% increase in placental lactogen secretion. The data suggest a major process of constitutive secretion and a minor role for regulated secretion from a storage pool.     

The contribution of SNAT1 to system A amino acid transporter activity in human placental trophoblast

System A-mediated amino acid transport across the placenta is important for the supply of neutral amino acids needed for fetal growth. All three system A subtypes (SNAT1, 2, and 4) are expressed in human placental trophoblast suggesting there is an important biological role for each. Placental system A activity increases as pregnancy progresses, coinciding with increased fetal nutrient demands. We have previously shown SNAT4-mediated system A activity is higher in first trimester than at term, suggesting that SNAT1 and/or SNAT2 are responsible for the increased system A activity later in gestation. However, the relative contribution of each subtype to transporter activity in trophoblast at term has yet to be evaluated. The purpose of this study was to identify the predominant subtype of system A in cytotrophoblast cells isolated from term placenta, maintained in culture for 66 h, by: (1) measuring mRNA expression of the three subtypes and determining the Michaelis-Menten constants for uptake of the system A-specific substrate, ¹⁴C-MeAIB, (2) investigating the contribution of SNAT1 to total system A activity using siRNA. Results: mRNA expression was highest for the SNAT1 subtype of system A. Kinetic analysis of ¹⁴C-MeAIB uptake revealed two distinct transport systems; system 1: K_m = 0.38 ± 0.12 mM, V_max = 27.8 ± 9.0 pmol/mg protein/20 min, which resembles that reported for SNAT1 and SNAT2 in other cell types, and system 2: K_m = 45.4 ± 25.0 mM, V_max = 1190 ± 291 pmol/mg protein/20 min, which potentially represents SNAT4. Successful knockdown of SNAT1 mRNA using target-specific siRNA significantly reduced system A activity (median 75% knockdown, n = 7). Conclusion: These data enhance our limited understanding of the relative importance of the system A subtypes for amino acid transport in human placental trophoblast by demonstrating that SNAT1 is a key contributor to system A activity at term.     

Interleukin-1 directly regulates hormone-dependent human breast cancer cell proliferation in vitro

Direct in vitro effects of IL-1 on hormone-dependent (MCF-7 and ZR-75-B) and independent (HS-578-T and MDA-231) human breast cancer cell proliferation were investigated in short-term and long-term cell cultures. For short-term (48 h) studies [3H]thymidine uptake was used as an index of proliferation, while for long-term (12 day) cultures actual cell numbers were determined. Initial studies, conducted with MCF-7 cells, demonstrated that both forms of recombinant human IL-1 (alpha and beta) at 10(-11) M inhibited [3H]thymidine uptake by MCF-7 by 70%, and by day 7 of the long-term study alpha and beta IL-1 at 10(-11) M inhibited MCF-7 cell growth by 80%. IL-1, while inhibiting the growth of another hormone-dependent breast cancer cell line; ZR-75-B, had no effect on the hormone-independent cell lines MDA-231 and HS-578-T. The differing proliferative responses of the hormone-dependent and independent cells to IL-1 may, in part, be due to the expression of IL-1 receptors on these cells, in that MCF-7 cells express IL-1 receptors [dissociation constant (Kd) = 2.0 x 10(-10) M; receptor density = 2,500 sites per cell and mol wt = 80,000] while the hormone-independent MDA-231 cells do not.     

Quantifying the syncytialisation of human placental trophoblast BeWo cells grown in vitro

We have generated lines of BeWo cells that constitutively and stably express either histone H2B tagged with the green fluorescent protein (GFP), or the mitochondrial targeting sequence of subunit VIII of cytochrome c oxidase fused with a red fluorescent protein; one line has nuclei that fluoresce green, the other mitochondria that fluoresce red. Expression of these tagged proteins has no effect on the rates of DNA, RNA and protein synthesis, or on the amounts of human chorionic gonadotropin (hCG) secreted after treatment with forskolin. We used fluorescence-activated cell sorting (FACS) to monitor the extent of cell fusion (syncytialisation) between these two lines; fused cells are readily and accurately detected by their green/red fluorescence. This assay should prove useful in the investigation of the molecular mechanisms involved in trophoblast syncytialisation.     

Purification of human placental trophoblast interferon by two-dimensional high performance liquid chromatography

Human placental trophoblast challenged with Sendai virus induced IFNs mainly of the beta-type (75%) and relatively low levels of the alpha-type (25%). A two-step high performance liquid chromatographic procedure ("two-dimensional HPLC") has been developed for the complete purification of the placental trophoblast interferon beta (tro-IFN-beta) from serum-containing culture supernatant. The method involved a combination of high performance liquid affinity chromatography (HPLAC) on Cibacron Blue 3GA immobilized on an activated pressure stable macroporous synthetic polymer, 2-hydroxyethyl methacrylate vinyl sulphone (HEMA-BIO 1000 VS), as the first dimension and reversed-phase high performance liquid chromatography (RP-HPLC) on Separon SGX C-18 as the second. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot experiments showed that the tro-IFN-beta was present as a 24 kDa protein. Densitometric scanning analysis of Coomassie-stained gel revealed the purity of the final preparation to be greater than 99%. The purified tro-IFN-beta had a specific activity of 1.03 x 10(8) IU/mg of protein and the overall recovery was 81% of the total IFN-beta activity in the crude preparation and 61% of the total IFN activity.     

Stem-cell-derived trophoblast organoids model human placental development and susceptibility to emerging pathogens

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Isolation and partial characterization of the basal cell membrane of human placental trophoblast

The function of the syncytiotrophoblast in maternal-fetal exchange is related to the properties of its microvillous (maternal-facing) and basal (fetal-facing) plasma membranes. We have previously reported the properties of the microvillous membrane (Smith, C.H., Nelson, D.M., King, B.F., Donohue, T.M., Ruzycki, S.M. and Kelley, L.K. (1977) Am. J. Obstet. Gynecol. 128, 190–196), and now describe the purification and partial characterization of the basal plasma membrane. Sonication and incubation with EDTA were used to isolate selectively the basal cell membrane. These steps were followed by a more conventional purification by centrifugation. The trophoblast was disrupted and its microvillous membrane and cytoplasmic contents were removed by sonication. The exposed basal cell membrane was selectively released from the underlying basal lamina by sonication in the presence of EDTA and further purified by discontinuous Ficoll gradient centrifugation. The material at the 4–10% Ficoll interface consisted of smooth membrane vesicles with internal microfilaments. It was 45-fold enriched in dihydroalprenolol binding activity and 11-fold enriched in ouabain binding activity. Other enzymatic analyses, including alkaline phosphatase, cytochrome-c oxidase, cytochrome-c reductase and galactosyl transferase indicated low contamination by other organelles. This procedure yields a preparation of relatively high purity which should be suitable for investigation of transport and other functions of the basal surface membrane of trophoblast. In principle, the purification procedures used may be applicable to other transporting epithelia.     

Morphologic and cytochemical identification of microperoxisomes in human and non-human primate placental trophoblast

Summary Human and non-human primate placental villi (near term) were incubated in an alkaline 3,3 diamino benzidine (DAB) medium to identify the distribution of microperoxisomes in these tissues. Catalase-positive microperoxisomes were present in small numbers in cytotrophoblast, but were virtually absent from syncytiotrophoblast. Microperoxisomes were roughly circular in shape and about 0.2–0.3 m in diameter. They always had a close association with an element of agranular endoplasmic reticulum. The function of microperoxisomes in placenta, as in many other tissues, is uncertain. What-ever their function, they are presumably more important in cytotrophoblast metabolism than in syncytium.     

Estrogen synthetase (aromatase) in cultured human term placental cells and neoplastic human trophoblast

Estrogen synthetase (aromatase) is present in large amounts in human term placenta. However, the localization of aromatase within the cellular structure of the placental villus is obscure. By immunocytochemical techniques using antibodies that separately recognize each component of the aromatase cytochrome P-450 enzyme system, the fraction of term placental trophoblast cells in primary culture expressing each aromatase component antigen increased from 20% in fresh mononucleated cells to about 65% for multinucleated giant cells after 72 h. In contrast, about 80% of human choriocarcinoma cells in continuous culture (JAr line) expressed each aromatase component antigen. The fraction of trophoblast cells in primary culture containing human chorionic gonadotropin increased from about 14% in fresh mononucleated cells to about 45% after 72 h and was about 30% in the choriocarcinoma cells. Fibroblast cells in culture, derived from trypsin-treated placental villi, contained aromatase activity, albeit much lower than term placental trophoblast cells. Aromatase specific activity in these placental fibroblasts did not change following growth with dibutyryl cAMP plus theophylline for 72 h.     

The effect of zinc on human trophoblast proliferation and oxidative stress

Adequate Zinc (Zn) intake is required to prevent multiple teratogenic effects however deviations from adequate Zn intake, including high maternal Zn status, have been linked to increased incidence of pregnancy complications, including those associated with inadequate placentation. Using placental trophoblast HTR8/SVneo cells and first trimester human placental explants (n = 12), we assessed the effects of varying Zn concentrations on trophoblast proliferation, viability, apoptosis and oxidative stress. Compared to physiologically normal Zn levels (20 µM), HTR-8/SVneo cell proliferation index was significantly lower in the presence of physiologically elevated (40 µM; P = .020) and supra-physiological (80 µM; P = .007) Zn. The latter was also associated with reduced proliferation (P = .004) and viability (P < .0001) in cultured placental explants, but not apoptosis. Reactive oxygen species production in HTR8/SVneo cultures was significantly higher in the presence of 80 µM Zn compared to all physiologically relevant levels. Oxidative stress, induced by an oxidizing agent menadione, was further exacerbated by high (80 µM) Zn. Zn did not affect lipid peroxidation in either HTR8/SVneo cells or placental explants or antioxidant defense mechanisms that included glutathione reductase and superoxide dismutase. Further study should focus on elucidating mechanisms behind impaired trophoblast proliferation and increased oxidative stress as a result of elevated Zn levels.     

Biochemical characterization and modulation of LH/CG-receptor during human trophoblast differentiation

Due to the key role of the human chorionic gonadotropin hormone (hCG) in placental development, the aim of this study was to characterize the human trophoblastic luteinizing hormone/chorionic gonadotropin receptor (LH/CG-R) and to investigate its expression using the in vitro model of human cytotrophoblast differentiation into syncytiotrophoblast. We confirmed by in situ immunochemistry and in cultured cells, that LH/CG-R is expressed in both villous cytotrophoblasts and syncytiotrophoblasts. However, LH/CG-R expression decreased during trophoblast fusion and differentiation, while the expression of hCG and hPL (specific markers of syncytiotrophoblast formation) increased. A decrease in LH/CG-R mRNA during trophoblast differentiation was observed by means of semi-quantitative RT-PCR with two sets of primers. A corresponding decrease ( approximately 60%) in LH/CG-R protein content was shown by Western-blot and immunoprecipitation experiments. The amount of the mature form of LH/CG-R, detected as a 90-kDa band specifically binding (125)I-hCG, was lower in syncytiotrophoblasts than in cytotrophoblasts. This was confirmed by Scatchard analysis of binding data on cultured cells. Maximum binding at the cell surface decreased from 3,511 to about 929 molecules/seeded cells with a kDa of 0.4-0.5 nM. Moreover, on stimulation by recombinant hCG, the syncytiotrophoblast produced less cyclic AMP than cytotrophoblasts, indicating that LH/CG-R expression is regulated during human villous trophoblast differentiation.     

Galectin-1 binds mucin in human trophoblast

Mucins are multifunctional highly glycosylated proteins expressed by the female reproductive tract. Differential expression of MUC1 and MUC15 has been shown in trophoblast. This study was undertaken to establish the distribution of mucin(s) in cytotrophoblast cell cultures using anti-bovine submaxillary mucin (BSM) and to investigate the possibility of MUC1/mucin(s) being a binding partner of trophoblast galectin-1. MUC1 is demonstrated here using immunocytochemistry on isolated cytotrophoblast and the HTR-8/SVneo extravillous trophoblast cell line but detection of additional trophoblast mucins cannot be excluded. Western blot analysis showed similar bands ranging from 30 to >200 kDa with anti-BSM and the well-known mucin antibodies HMFG1 and B72.3. Immunocytochemistry and cell-based ELISA data were found to support that all of the antibodies used are reactive with BSM, suggesting the presence of shared epitopes between BSM and trophoblast mucin(s). Binding of galectin-1 to trophoblast MUC1/mucin(s) was analyzed using a solid-phase assay and co-immunoprecipitation. Recombinant galectin-1 binding to isolated trophoblast mucin in solid-phase assay was sensitive to lactose, a carbohydrate inhibitor of galectin binding. In whole HTR-8/SVneo lysates, ~200 kDa mucin was detected in galectin-1 immunoprecipitates, while endogenous galectin-1 was present in BSM-immunoprecipitates. Furthermore, double fluorescence immunocytochemistry showed overlap of galectin-1 and trophoblast mucins at the plasma membrane of HTR-8/SVneo cells. These results suggest that trophoblast mucin(s) could act as binding partners of galectin-1, in a carbohydrate-dependent manner.     

Hemeoxygenase expression in human placenta and placental bed implies a role in regulation of trophoblast invasion and placental function.

The purpose of this study was to examine the expression of hemeoxygenases HO-1 and HO-2, which are responsible for the production of carbon monoxide (CO), in the human placenta and placental bed and to determine the role of inhibitors of HO on placental perfusion pressure. We hypothesized that HO is expressed within the placenta and that invading cytotrophoblast cells (CTB) express HO isoforms. The expression of HO-1 and HO-2 was studied on placenta and placental bed biopsies, obtained using a transcervical sampling technique, from normal human pregnancies between 8 and 19 wk gestation and at term. In the placenta, HO-2 immunostaining was prominent in syncytiotrophoblast in the first trimester and reduced toward term (P<0.0005). HO-2 endothelial immunostaining was weak in the first trimester, but increased by term (P<0.0005). Within the placental bed, HO-2 was expressed by CTB in cell columns, the cytotrophoblast shell, and cell islands. Both intravascular CTB and interstitial CTB expressed HO-2. HO-1 immunostaining was low in the placenta but intense on the CTB within the placental bed. A striking feature was the absence of HO-1 from the proximal layers of cell columns, with strong expression on the more distal CTB layers of the cell columns. In placental perfusion studies, a significant dose-dependent increase in perfusion pressure was observed in the presence of zinc protoporphyrin, an inhibitor of HO. These results suggest a role for CO in placental function, trophoblast invasion, and spiral artery transformation. Hemeoxygenase expression in human placenta and placental bed implies a role in regulation of trophoblast invasion and placental function.