Oxygen-Sensitive K+ Channels Modulate Human Chorionic Gonadotropin Secretion from Human Placental Trophoblast

Human chorionic gonadotropin (hCG) is a key autocrine/paracrine regulator of placental syncytiotrophoblast, the transport epithelium of the human placenta. Syncytiotrophoblast hCG secretion is modulated by the partial pressure of oxygen (pO₂), reactive oxygen species (ROS) and potassium (K⁺) channels. Here we test the hypothesis that K⁺ channels mediate the effects of pO₂ and ROS on hCG secretion. Placental villous explants from normal term pregnancies were cultured for 6 days at 6% (normoxia), 21% (hyperoxia) or 1% (hypoxia) pO₂. On days 3–5, explants were treated with 5mM 4-aminopyridine (4-AP) or tetraethylammonium (TEA), blockers of pO₂-sensitive voltage-gated K⁺ (K_V) channels, or ROS (10–1000μM H₂O₂). hCG secretion and lactate dehydrogenase (LDH) release, a marker of necrosis, were determined daily. At day 6, hCG and LDH were measured in tissue lysate and ⁸⁶Rb (K⁺) efflux assessed to estimate syncytiotrophoblast K⁺ permeability. hCG secretion and ⁸⁶Rb efflux were significantly greater in explants maintained in 21% pO₂ than normoxia. 4-AP/TEA inhibited hCG secretion to a greater extent at 21% than 6% and 1% pO₂, and reduced ⁸⁶Rb efflux at 21% but not 6% pO₂. LDH release and tissue LDH/hCG were similar in 6%, 21% and 1% pO₂ and unaffected by 4-AP/TEA. H₂O₂ stimulated ⁸⁶Rb efflux and hCG secretion at normoxia but decreased ⁸⁶Rb efflux, without affecting hCG secretion, at 21% pO₂. 4-AP/TEA-sensitive K⁺ channels participate in pO₂-sensitive hCG secretion from syncytiotrophoblast. ROS effects on both hCG secretion and ⁸⁶Rb efflux are pO₂-dependent but causal links between the two remain to be established.     

Purification of Human Placental Trophoblast Interferon by Two-Dimensional High Performance Liquid Chromatography

Abstract

Human placental trophoblast challenged with Sendai virus induced IFNs mainly of the β-type (75%) and relatively low levels of the α-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-β) 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-β 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-β had a specific activity of 1.03 × 10⁸ IU/mg of protein and the overall recovery was 81% of the total IFN-β activity in the crude preparation and 61% of the total IFN activity.     

The Human Placental Sexome Differs between Trophoblast Epithelium and Villous Vessel Endothelium

Molecular mechanisms underlying sexual dimorphism in mammals, fetal sex influences on intrauterine development, and the sex-biased susceptibility for selected diseases in adulthood are novel areas of current research. As importantly, two decades of multifaceted research has established that susceptibility to many adult disorders originates in utero, commonly secondary to the effects of placental dysfunction. We hypothesized that fetal sex influences gene expression and produces functional differences in human placentas. We thus extended previous studies on sexual dimorphism in mammals, which used RNA isolated from whole tissues, to investigate the effects of sex on four cell-phenotypes within a single key tissue, human placental villi. The cells studied included cytotrophoblasts, syncytiotrophoblast, arterial and venous endothelial cells. The cells were isolated from placentas of male or female fetuses and subjected to microarray analysis. We found that fetal sex differentially affected gene expression in a cell-phenotype dependent manner among all four cell-phenotypes. The markedly enriched pathways in males were identified to be signaling pathways for graft-versus-host disease as well as the immune and inflammatory systems that parallel the reported poorer outcome of male fetuses. Our study is the first to compare global gene expression by microarray analysis in purified, characterized, somatic cells from a single human tissue, i.e. placental villi. Importantly, our findings demonstrate that there are cell-phenotype specific, and tissue-specific, sex-biased responses in the human placenta, suggesting fetal sex should be considered as an independent variable in gene expression analysis of human placental villi.     

Regulation of Human Trophoblast GLUT1 Glucose Transporter by Insulin-Like Growth Factor I (IGF-I)

Glucose transport to the fetus across the placenta takes place via glucose transporters in the opposing faces of the barrier layer, the microvillous and basal membranes of the syncytiotrophoblast. While basal membrane content of the GLUT1 glucose transporter appears to be the rate-limiting step in transplacental transport, the factors regulating transporter expression and activity are largely unknown. In view of the many studies showing an association between IGF-I and fetal growth, we investigated the effects of IGF-I on placental glucose transport and GLUT1 transporter expression. Treatment of BeWo choriocarcinoma cells with IGF-I increased cellular GLUT1 protein. There was increased basolateral (but not microvillous) uptake of glucose and increased transepithelial transport of glucose across the BeWo monolayer. Primary syncytial cells treated with IGF-I also demonstrated an increase in GLUT1 protein. Term placental explants treated with IGF-I showed an increase in syncytial basal membrane GLUT1 but microvillous membrane GLUT1 was not affected. The placental dual perfusion model was used to assess the effects of fetally perfused IGF-I on transplacental glucose transport and syncytial GLUT1 content. In control perfusions there was a decrease in transplacental glucose transport over the course of the perfusion, whereas in tissues perfused with IGF-I through the fetal circulation there was no change. Syncytial basal membranes from IGF-I perfused tissues showed an increase in GLUT1 content. These results demonstrate that IGF-I, whether acting via microvillous or basal membrane receptors, increases the basal membrane content of GLUT1 and up-regulates basal membrane transport of glucose, leading to increased transepithelial glucose transport. These observations provide a partial explanation for the mechanism by which IGF-I controls nutrient supply in the regulation of fetal growth.     

Schistosoma japonicum Soluble Egg Antigens Attenuate Invasion in a First Trimester Human Placental Trophoblast Model

Background

Schistosomiasis affects nearly 40 million women of reproductive age, and is known to elicit a pro-inflammatory signature in the placenta. We have previously shown that antigens from schistosome eggs can elicit pro-inflammatory cytokine production from trophoblast cells specifically; however, the influence of these antigens on other characteristics of trophoblast function, particularly as it pertains to placentation in early gestation, is unknown. We therefore sought to determine the impact of schistosome antigens on key characteristics of first trimester trophoblast cells, including migration and invasion.

Methods

First trimester HTR8/SVneo trophoblast cells were co-cultured with plasma from pregnant women with and without schistosomiasis or schistosome soluble egg antigens (SEA) and measured cytokine, cellular migration, and invasion responses.

Results

Exposure of HTR8 cells to SEA resulted in a pro-inflammatory, anti-invasive signature, characterized by increased pro-inflammatory cytokines (IL-6, IL-8, MCP-1) and TIMP-1. Additionally, these cells displayed 62% decreased migration and 2.7-fold decreased invasion in vitro after treatment with SEA. These results are supported by increased IL-6 and IL-8 in the culture media of HTR8 cells exposed to plasma from Schistosoma japonica infected pregnant women.

Conclusions

Soluble egg antigens found in circulation during schistosome infection increase pro-inflammatory cytokine production and inhibit the mobility and invasive characteristics of the first trimester HTR8/SVneo trophoblast cell line. This is the first study to assess the impact of schistosome soluble egg antigens on the behavior of an extravillous trophoblast model and suggests that schistosomiasis in the pre-pregnancy period may adversely impact placentation and the subsequent health of the mother and newborn.     

瘦蛋白介导的人食欲与体重调节机制

瘦蛋白(leptin)介导了一个由5个基因的表达与调控组成的食欲调节系统,从而实现了人体重的反馈调节。深入分析类体重调节的,矿子机制对于阐明肥胖发生的生理学机制,以及对于抗肥胖药物的设计和筛选具育重要意义。     

Derivation of Human Trophoblast Stem Cells

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Human Trophoblast Cells Are Permissive to the Complete Replicative Cycle of Human Cytomegalovirus

Human trophoblast cells were permissively infected by human cytomegalovirus. The kinetics of viral immediate-early, early, and late gene expression was clearly delayed compared to that in fibroblasts. Productive infection was unequivocally proven by the detection of virion particles, infectious virus in trophoblast culture supernatant, and cell-to-cell spread of cytomegalovirus from infected trophoblasts to uninfected fibroblasts. These observations indicate that infected trophoblasts may be involved in maternofetal transmission of human cytomegalovirus.     

Sp1和Sp3介导的转录调控

基本转录因子Sp1和Sp3对转录调控区GC盒有很强的亲和力,参与几乎所有细胞功能,包括细胞增殖、凋亡、分化和新生物的转化.但在同一细胞中Sp1和Sp3对不同基因的作用并不相同,二者对基因特异性的转录调控是Sp1和Sp3研究领域的重要问题.近年来发现,Sp1和Sp3自身表达水平、结合的靶序列、磷酸化、糖基化等翻译后修饰,其他蛋白质的结合以及染色质结构与修饰等方面均可影响Sp1和Sp3的转录活性.本文从Sp1和Sp3蛋白参与转录调节的机制以及影响其基因特异性转录活性的诸方面因素这两大侧面,介绍了近年来的最新进展.     

Amino-acid Sequence of Human Placental Lactogen

AN earlier report from this laboratory¹ described striking homologies in the amino-acid composition of the peptides obtained from trypsin cleavage of human placental lactogen (HPL) and human growth hormone (HGH) and established a firm biochemical basis for the similarities in biological and immunological activity of the two hormones. The intrachain disulphide bonds were placed in similar locations and the carboxyl-terminal amino-acid sequences were shown to be identical except for one out of fourteen residues². In other studies^3,4, the amino-terminal sequence was determined and noted to be homologous for eleven of the first seventeen residues. We now report the complete amino-acid sequence of HPL and a comparison with the revised structure for HGH recently reported⁴. These two protein hormones are strikingly similar in structure, being identical in 85% of the corresponding positions. Of twenty-eight amino-acid substitutions, twenty-five are favoured changes and all but two of the twenty-five can be explained in terms of a single base change in the triplet codon⁵.     

Alpha-1-Antitrypsin: A Novel Human High Temperature Requirement Protease A1 (HTRA1) Substrate in Human Placental Tissue

The human serine protease high temperature requirement A1 (HTRA1) is highly expressed in the placental tissue, especially in the last trimester of gestation. This suggests that HTRA1 is involved in placental formation and function. With the aim of a better understanding of the role of HTRA1 in the placenta, candidate substrates were screened in a placenta protein extract using a gel-based mass spectrometric approach. Protease inhibitor alpha-1-antitrypsin, actin cytoplasmic 1, tropomyosin beta chain and ten further proteins were identified as candidate substrates of HTRA1. Among the identified candidate substrates, alpha-1-antitrypsin (A1AT) was considered to be of particular interest because of its important role as protease inhibitor. For investigation of alpha-1-antitrypsin as substrate of HTRA1 synthetic peptides covering parts of the sequence of alpha-1-antitrypsin were incubated with HTRA1. By mass spectrometry a specific cleavage site was identified after met-382 (AIPM³⁸²↓³⁸³SIPP) within the reactive centre loop of alpha-1-antitrypsin, resulting in a C-terminal peptide comprising 36 amino acids. Proteolytic removal of this peptide from alpha-1-antitrypsin results in a loss of its inhibitor function. Beside placental alpha-1-antitrypsin the circulating form in human plasma was also significantly degraded by HTRA1. Taken together, our data suggest a link between the candidate substrates alpha-1-antitrypsin and the function of HTRA1 in the placenta in the syncytiotrophoblast, the cell layer attending to maternal blood in the villous tree of the human placenta. Data deposition: Mass spectrometry (MS) data have been deposited to the ProteomeXchange with identifier PXD000473.