Lucie Simoneau

ERK1/2 and p38 regulate trophoblasts differentiation in human term placenta

Mitogen‐activated protein kinases (MAPKs) control many cellular events from complex programmes, such as embryogenesis, cell differentiation and proliferation, and cell death, to short‐term changes required for homeostasis and acute hormonal responses. However, little is known about expression and activation of classical MAPKs, extracellular signal‐regulated kinase1/2 (ERK1/2) and p38 in human placenta. Therefore, we examined the expression of ERK1/2 and p38 in trophoblasts from human term placenta, and their implication in differentiation. In vitro, freshly isolated cytotrophoblast cells, cultivated in 10% fetal bovine serum (FBS), spontaneously aggregate and fuse to form multinucleated cells that phenotypically resemble mature syncytiotrophoblasts, that concomitantly produce human chorionic gonadotropin (hCG) and human placental lactogen (hPL). This study shows that the level of ERK1/2 and p38 decreases with increasing days of culture, to reach an undetectable level after 5 days of culture. Moreover, pretreatment of cells with an ERK1/2‐specific inhibitor (PD98059) and/or a p38‐specific inhibitor (SB203580) suppressed trophoblast differentiation. Our results also demonstrate that the p38 pathway is highly solicited as compared to the ERK1/2 pathway in the differentiation process. Furthermore, ERK1/2 and p38 are rapidly activated upon addition of FBS, but the activation of p38 is delayed compared to that of ERK1/2. In summary, this study showed that ERK1/2 and p38 pathways are essential to mediate initiation of trophoblast differentiation.

Calcium uptake and calcium transporter expression by trophoblast cells from human term placenta

Placental transfer of maternal calcium (Ca(2+)) is a crucial step for fetal development although the biochemical mechanisms responsible for this process are largely unknown. This process is carried out in vivo by the placental syncytiotrophoblast layer. The aim of this study was to define the membrane gates responsible for the syncytiotrophoblast Ca(2+) entry, the first step in transplacental transfer. We have investigated the basal Ca(2+) uptake by primary culture of human term placenta syncytiotrophoblast. Kinetic studies revealed an active extracellular Ca(2+) uptake by cultured human syncytiotrophoblast. We demonstrated by Northern blot the presence of transcript for calcium transporter type 1 (CaT1) in cultured human syncytiotrophoblast and CaT1 expression was further confirmed by reverse transcription polymerase chain reaction (RT-PCR). In addition, the expression of calcium transporter type 2 (CaT2) was revealed by RT-PCR in cultured human syncytiotrophoblast. It has been reported that the activity of this family of Ca(2+) channels is voltage-independent, and is not sensitive to L-type Ca(2+) channels agonist and antagonist. Interestingly, modulation of membrane potential by extracellular high potassium concentration and valinomycin had no effect on the basal Ca(2+) uptake of human syncytiotrophoblast. Moreover, the addition of L-type Ca(2+) channel modulators (Bay K 8644 and nitrendipine) to the incubation medium had also no effect on the basal Ca(2+) uptake, suggesting that the process is mainly voltage-independent and does not involved L-type Ca(2+) channels. On the other hand, we observed that two known blockers of CaT-mediated Ca(2+) transport, namely extracellular magnesium (Mg(2+)) and ruthenium red, dose-dependently inhibited Ca(2+) uptake by cultured human syncytiotrophoblast. Therefore, our results suggest that basal Ca(2+) uptake of human syncytiotrophoblast may be assured by CaT1 and CaT2.

Expression of Calcium Channels along the Differentiation of Cultured Trophoblast Cells from Human Term Placenta

Abstract Placental transfer of maternal calcium (Ca2+) is carried out in vivo by the syncytiotrophoblast layer. Although this process is crucial for fetal development, it remains poorly understood. Cytotrophoblasts isolated from human term placenta undergo spontaneous syncytiotrophoblast-like morphological and biochemical differentiation in vitro and are thought to reflect in vivo syncytiotrophoblast. In the present study, we characterized the Ca2+ uptake potential and the expression of several Ca2+ channels by human trophoblasts during differentiation in vitro for up to 6 days. Secretion of hCG (specific differentiation marker) and uptake of Ca2+ by trophoblasts increased gradually as a function of days in culture. Both hCG secretion and Ca2+ uptake were maximal on Day 4 and declined on Days 5–6. Expression of the Ca2+ transporter proteins CaT1 and CaT2 was revealed by reverse transcription-polymerase chain reaction in cytotrophoblasts freshly isolated from human term placenta. In addition, messengers for two L-type Ca2+ channel isoforms (α1C and α1D) were also detected. Levels of CaT1, CaT2, and L-type Ca2+ channel mRNA increased gradually during culture, reaching a maximum between Days 2 and 3. In contrast to CaT1 and CaT2 expression that declined thereafter to levels observed on Day 1, L-type channel expression decreased by 50% but remained above the expression level of Day 1. Our results indicate that the pattern of CaT1 and CaT2 expression correlates with the Ca2+ uptake potential along the differentiation of cultured human trophoblasts isolated from term placenta. This correlation provides circumstantial evidence for a role of this family of channels in basal Ca2+ uptake by the syncytiotrophoblast.

Impact of a cholesterol enriched diet on maternal and fetal plasma lipids and fetal deposition in pregnant rabbits.

Pregnancy is associated with a hypercholesterolemic and a hyperlipidemic state. The totality of the essential fatty acids and 50% of the lipids needed by the fetus are transferred by the placenta from the maternal circulation. The hypothesis of this study is that an augmentation of the maternal plasmatic cholesterol is modifying the fetal lipids accumulation and development during rabbit pregnancy. To demonstrate the impact of a cholesterol enriched diet on plasma lipids during rabbits pregnancy and on their fetus, we have established two groups: control and hypercholesterolemic rabbits (fed with a 0.2% cholesterol diet). Blood samples were collected before mating and at each trimester of pregnancy for analysis of lipid fractions and their lipoproteins. Plasma analysis shows that starting the 10th day of pregnancy the concentration of total-cholesterol and lipoproteins decreases for both groups. We have demonstrated that for the hypercholesterolemic group, concentrations of total-cholesterol (631%) and lipoproteins are significantly higher at the end of pregnancy than those for the control group. For both groups, after 20 days of pregnancy, triglycerides metabolism was biphasic showing a significant increase followed by a diminution in their concentration. In both groups, free fatty acids increases significantly at the end of the pregnancy (537.5% for the control group and 462.5% for the hypercholesterolemic group). Furthermore, the offsprings of hypercholesterolemic dams manifest a lower birth weight (15.5%) than those of control group. Our results demonstrate that a cholesterol enriched diet modifies greatly the fetal development and lipid metabolism during rabbits pregnancy. These modifications could be useful for the understanding of the interaction between diet and fetal development in rabbit and probably during human pregnancy.

Calcium-binding proteins : Distribution and implication in mammalian placenta ()

During gestation, transport by the placenta is solely responsible for nutrient supply to the developing fetus. In this context, calcium (Ca2+) transport machinery of the placenta thus represents the primary tissue site for regulating fetal Ca2+ homeostasis. In humans, the transplacental movements of Ca2+ increase dramatically during the last trimester of gestation, when fetal skeletal mineralization is at its highest. However, little is known about the exact mechanism of transport. Evidence suggests that some developmentally expressed cytosolic Ca2+-binding proteins (CaBPs) have an important role in regulating or shuttling cytosolic Ca2+ since they are endowed with a high affinity for Ca2+ (∼106M−1). CaBPs belong to a large family of eukaryotic proteins containing a specific helix-loop-helix structure, referred to as the EF-hand motif, which counts more than 200 members. Several of these CaBPs were identified in the placenta: CaBP9k, CaBP28k, CaBP57k, oncomodulin, S-100P, S-100α, and S-100β. This review discusses the current views in this field to guide future investigations into the localization and functions of CaBPs during Ca2+ intracellular homeostasis in the placenta.

Calcitonin receptor in human placental syncytiotrophoblast brush border and basal plasma membranes

Abstract The physiology of calcium transport through the placenta has not been studied thoroughly. In particular, the effect of calcaemic hormones on this process has never been reported. In this paper we questioned if calcitonin, a hypocalcaemic hormone, is also implicated in the regulation of calcium transport by one of the placental syncytiotrophoblast bipolar membranes. In order to investigate the implication of calcitonin on calcium transport, we first studied whether this hormone binds to any of these bipolar membranes i.e. purified syncytiotrophoblast brush border (facing the mother) and basal plasma membranes (facing the fetus). The initiation of binding of human [ 125 I]calcitonin to the two types of membranes was rapid and reached a steady state after 10 min of incubation at 37°C. The number of binding sites and the affinity of these receptors for the hormone were studied for each type of membrane, with concentrations of [ 125 I]calcitonin varying from 0.01 to 1.8 nM. Scatchard analysis revealed a single affinity binding site for human calcitonin with K d s of 0.83 ± 0.09 nM and 0.67 ± 0.26 nM for brush border and basal plasma membranes respectively. The maximal number of receptors was significantly different ( p B max of 66.64 ± 9.15 fmol/mg protein for brush border membranes and 19.66 ± 2.80 fmol/mg protein for basal plasma membranes. Competitive displacement of [ 125 I]calcitonin with other ligands showed the following potencies between human calcitonin > salmon calcitonin > calcitonin gene-related peptides and segments analogues but no competition with some human calcitonin gene-related peptides fragments. Half-maximal displacement concentration for human calcitonin was reached at approximatively 1 nM for BBM and 0.1 nM for BPM. Calcitonin stimulated inositol phosphate production in both membranes by 175% and 330% in BBM and BPM, respectively. We conclude that calcitonin receptors are present in the two polar syncytiotrophoblast membranes, but are more abundant in brush border membranes, facing the mother. These results also suggest that in the placenta, both maternal and fetal calcitonin might intervene in ion transport and particularly calcium transport or on the intracellular metabolism of the placental syncytiotrophoblast cells.

Expression of Calbindin-D28k (CaBP28k) in Trophoblasts from Human Term Placenta

Abstract Calbindin-D28k (CaBP28k) belongs to a large class of eucaryotic proteins that bind calcium (Ca2+) to a specific helix-loop-helix structure. To date, this protein was mainly linked to brain, kidneys, and pancreas. Here, we demonstrate for the first time the existence of CaBP8k in the human placental trophoblasts of the human term placenta. Placental Ca2+ transfer from maternal to fetus is crucial for fetal development, although the biochemical mechanisms responsible for this process are largely unknown. In the current study, we have investigated the 45Ca2+ uptake by human trophoblast cells in correlation with the expression CaBP28k. The expression of CaBP28k was determined by Northern blot analysis, reverse transcriptase polymerase chain reaction (RT-PCR), immunochemistry, and Western blot analysis. Indeed, Northern blot analysis revealed the presence of a CaBP28k transcript in syncytiotrophoblasts, cytotrophoblast cells, and HEK-293 cells. This was further confirmed by RT-PCR analysis followed by sequencing. In addition, anti-CaBP28k labeling was associated with cytotrophoblast and syncytiotrophoblast tissues in placental tissue sections and in vitro cultured cells. The presence of CaBP28k protein in these cells was confirmed by Western blotting. Cytotrophoblast cells isolated from human term placenta showed differentiation into syncytiotrophoblasts in culture according to the increase in hCG secretion. Both Ca2+ uptake and hCG secretion by trophoblasts increased gradually and were high at Day 4. Taken together, these data suggest that CaBP28k may play a role in Ca2+ transport or cell development in human trophoblast possibly trough Ca2+ buffering.

Calbindin-D9k (CaBP9k) localization and levels of expression in trophoblast cells from human term placenta.

During pregnancy, the calcium (Ca2+) transport machinery of the placenta is solely responsible for the nutrient supply to the developing fetus, where active Ca2+ transport occurs from the mother to the fetus. As part of a larger study to determine the role of Ca2+ in placental transport in vivo, we questioned whether calbindin-D9k (CaBP9k), which is mainly expressed in duodenum, uterus, and placenta of several mammals, is present in cytotrophoblast cells and syncytiotrophoblasts of human term placenta. We were interested in this protein because of its potential importance in serving as an indicator of Ca2+ availability and utilization in the placenta. Here, we demonstrated that CaBP9k transcript is present in both cell types, with a lower expression in cytotrophoblast cells as compared to syncytiotrophoblasts. Moreover, we showed by immunochemistry that CaBP9k protein was present in cytotrophoblast and syncytiotrophoblast placental tissue sections as well as in cultured cells. The occurrence of CaBP9k protein in trophoblast cells was further confirmed by Western blot analysis. Thus, these results indicate for the first time that CaBP9k is unequivocally expressed by trophoblast cells from human term placenta.

Calcium homeostasis in human placenta: role of calcium-handling proteins.

The human placenta is a transitory organ, representing during pregnancy the unique connection between the mother and her fetus. The syncytiotrophoblast represents the specialized unit in the placenta that is directly involved in fetal nutrition, mainly involving essential nutrients, such as lipids, amino acids, and calcium. This ion is of particular interest since it is actively transported by the placenta throughout pregnancy and is associated with many roles during intrauterine life. At term, the human fetus has accumulated about 25-30 g of calcium. This transfer allows adequate fetal growth and development, since calcium is vital for fetal skeleton mineralization and many cellular functions, such as signal transduction, neurotransmitter release, and cellular growth. Thus, there are many proteins involved in calcium homeostasis in the human placenta.

Differential Expression of Membrane and Soluble Adenylyl Cyclase Isoforms in Cytotrophoblast Cells and Syncytiotrophoblasts of Human Placenta

Adenylyl cyclase (AC) activity is ubiquitous in mammalian cells, and various forms of this enzyme exist that widely differ with regard to tissue distribution, abundance, and modes of regulation. Human placenta is made, among others, of cytotrophoblast cells and syncytiotrophoblasts. This latter is a polynucleate structure that originates from the differentiation of proliferative mononucleated cytotrophoblast cells, the placental stem cell, into syncytiotrophoblasts. In vitro, this differentiation process is associated with a concomitant increase in cellular levels of cAMP and enhanced expression of genes representative of syncytiotrophoblasts endocrine activity. Thus, in this study we evaluated the differential distribution of AC isoforms in cytotrophoblast cells and syncytiotrophoblasts by reverse transcription-polymerase chain reaction (RT-PCR) using total RNA or purified mRNA. Our results demonstrate that all membrane and soluble AC mRNA isoforms are present in both cell types. Interestingly in syncytiotrophoblasts, AC4 and AC8 mRNA are highly expressed, while AC1, AC2 mRNA are less abundant when compared to cytotrophoblast cells. Additionally, the soluble AC is expressed in both trophoblast cells, but its expression is greatly reduced in differentiated cells, syncytiotrophoblasts. The presence of these AC proteins in both cells was confirmed by Western blotting. Taken together, these data help us to characterize the different AC isoforms in human cytotrophoblast cells and syncytiotrophoblasts, and demonstrate that the AC isoforms expression seems to be mainly modulated in groups 1 and 2. Moreover, the important decrease of the soluble AC isoform in syncytiotrophoblasts as compared to cytotrophoblast cells could suggest an important role of this AC in the extravillous trophoblast formation.