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.

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.

Expression of cFABP and PPAR in trophoblast cells: effect of PPAR ligands on linoleic acid uptake and differentiation.

Throughout gestation, fetal growth depends, in part, on placental transfer of maternal essential fatty acid (EFA) and long-chain polyunsaturated fatty acid. All fatty acid (FA) can cross lipid bilayer by simple diffusion, such as those in the syncytiotrophoblasts, the multinucleated, terminally differentiated trophoblast cells. The trophoblasts differentiation process is accompanied by an increase of human chorionic gonadotropin (hCG) secretion and an inhibition of Human Achaete-Scute Homologue-2 expression (Hash-2). Furthermore, a number of FA-binding proteins (FABPs) have been identified in membrane and cytoplasm of mammalian cells, which are thought to facilitate the transfer of FA across membranes and their intracellular channeling. Thus, the aim of this study was to investigate the implication of cFABPs in linoleic acid (LA) uptake by human trophoblast cells according to differentiation. Moreover, since peroxisome proliferator-activated receptor (PPARs) regulate the expression of cFABP and play an important role in trophoblast cells differentiation, the effects of PPARs ligands are verified on cFABP expression and differentiation. Herein, we reported the increase of the expression of liver and heart FABP (L- and H-FABP) upon differentiation of trophoblast cells, an inhibition of PPAR alpha and beta, while PPAR gamma levels remains unchanged. The nonselective peroxisome-proliferating agents, bezafibrate and LA, impaired trophoblast differentiation, and reduced L- and H-FABP expression. Furthermore, cobalt, a chemical agent known to mimic hypoxia, inhibits trophoblast cells differentiation and diminishes H-, L-FABP and PPARs expression. Finally, both treatments show no influence on LA uptake by trophoblast cells. In conclusion, this study showed that there is no correlation between the expression of H- and L-FABP and LA uptake by trophoblast cells and that bezafibrate and LA greatly impaired trophoblast cells differentiation.

Calcium channels, transporters and exchangers in placenta: a review.

Calcium (Ca2+) entry in cells is crucial for development and physiology of virtually all cell types. It acts as an intracellular (second) messenger to regulate a diverse array of cellular functions, from cell division and differentiation to cell death. Among candidates for Ca2+ entry in cells are-voltage-dependant Ca2+ channels (VDCCs), transient receptor potential (TRP)-related Ca2+ channels and store-operated Ca2+ (SOC) channels. Plasma membrane Ca2+-ATPases (PMCA) and Na+/Ca2+ exchanger (NCX) are mainly responsible for Ca2+ extrusion. These different Ca2+channels/transporters and exchangers exhibit specific distribution and physiological properties. During pregnancy, the syncytiotrophoblast layer of the human placenta transfers as much as 30 g of Ca2+ from the mother to the fetus, especially in late gestation where Ca2+ transport through different channels must increase in response to the demands of accelerating bone mineralization of the fetus. The identification and characterization of the different Ca2+ channels/transporters and exchangers on the brush-border membrane (BBM) facing the maternal circulation, and the basal plasma membrane (BPM) facing the fetal circulation; placental membrane of the syncytiotrophoblasts have been the focus of numerous studies. This review discusses current views in this field regarding localization and functions during transcellular Ca2+ entry and extrusion from cells particularly in the placenta.

Evaluation of 3-hydroxy-3-methylglutaryl-COA-reductase, cholesterol-7alpha-hydroxylase and acyl-COA:cholesterol acyltransferase activities: alternative chromatographic methods to separate metabolites.

Alternative HPLC and solid-phase extraction column methods were developed to separate metabolites of enzymes involved in cholesterol metabolism in rabbit liver microsomes: hydroxyl-methylglutaryl-CoA reductase, cholesterol-7alpha-hydroxylase and acyl-CoA:cholesterol acyltransferase. A comparison method of thin-layer chromatography and solid-phase extraction column were assayed to separate substrate and metabolite of hydroxy-methylglutaryl-CoA reductase, whereas for cholesterol-7alpha-hydroxylase and acyl-CoA:cholesterol acyltransferase, this comparison was done between thin layer chromatography and HPLC. The results obtained by the new analytical chromatographic methods are not significantly different than those observed in literature. Moreover a larger percentage recovery was obtained for analysed metabolites. Our results demonstrate the reliability of these alternative chromatographic techniques and showed that they are valuable tools to precisely and rapidly measure the activity of those enzymes.

Influence of a maternal cholesterol-enriched diet on [1-14C]-linoleic acid and L-[4, 5-3H]-leucine entry in plasma of rabbit offspring.

Fetal development requires an important entry of essential free fatty acids (EFFA) and essential amino acids (EAA) into the fetal circulation. We have reported that a 0.2% enriched-cholesterol diet (ECD) during rabbit gestation significantly reduces fetus weight compared to control diet. It is known that dietary linoleic acid deficiency, an EFFA, during the fetal development induces an important impair to the somatic development. Moreover, intrauterine growth retardation induced a reduction of the flux of leucine, an EAA, from maternal to fetal circulation. Therefore, we hypothesized that the administration of an ECD induces modifications of placental lipid composition concomitant alterations of the transfer of linoleic acid and leucine in fetal circulation. Quantification of placental lipids revealed that in the ECD group a reduction of total-cholesterol (TC) and free-cholesterol (FC) is observed, however an increased in FFA and phospholipids is noticed when compared to the control group. In placenta from the ECD group, the FC/ TC ratio is significantly reduced compared to the control group. In the ECD group, the liver shows an increase of TC, FC and FFA compared to the control group. However, the quantity of triacylglycerol present in the liver from the ECD is significantly reduced compared to the control group. To evaluate the placental transfer of some essential nutrients, intravenous injection of [1-14C]-linoleic acid or L-[4, 5-3H]-leucine to term rabbit (control and ECD group) were done. Two hours later, rabbits were euthanized and we collected placenta, livers and blood from dams and offspring. The concentrations of both radiolabeled molecules (linoleic acid and its esterified form or leucine) were higher in the plasma of ECD offspring than those found in offspring from control diet. Despite such alteration of placental lipid composition, linoleic acid and leucine transfer by the placenta was not compromised but rather increased.

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

During pregnancy, the calcium (Ca(2+)) transport machinery of the placenta is solely responsible for the nutrient supply to the developing fetus, where active Ca(2+) transport occurs from the mother to the fetus. As part of a larger study to determine the role of Ca(2+) 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 Ca(2+) 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.

Effects of low concentrations of organochlorine compounds in women on calcium transfer in human placental syncytiotrophoblast.

For most Canadians, food represents one of the major sources of environmental contaminants. Among them, organochlorine compounds (OCs) are known to affect calcium (Ca2+) homeostasis. They are neurotoxic by perturbation of Ca2+ channels and pumps, and they interfere with protein kinase C (PKC) and Ca2+ binding protein (CaBP). Ca2+ is an essential element to adequate fetal growth and development. The aim of the present study is to determine the relation between low environmental maternal exposure to OCs, such as polychlorinated biphenyls (PCB 153), Aroclor 1260, p,p'-dichlorodiphenyltrichloroethane (DDT) and p,p'-dichlorodiphenyl-dichloroethane (DDE), Ca2+ levels in serum and placenta, placental Ca2+ transfer, and newborn development. Total Ca2+ and OCs were measured in women's serum samples, as well as in umbilical cord's serum and placenta at term. Placentas were taken for trophoblast cells isolation and Ca2+ incorporation kinetic experiments. Our results were obtained from 30 pregnant women from the southwestern area of Quebec. Concentrations of Aroclor 1260, PCB 153, DDE, and DDT were respectively 6.1, 6.0, 3.1, and 2.9 times lower in the umbilical cord serum than in the mother's serum at term. In the placenta, DDE was accumulated at higher levels than other contaminants. A tendency towards an inverse relation was observed for in OCs found in three compartments and Ca2+ levels in maternal serum and in placental tissues. Maternal Ca2+ concentrations do not influence Ca2+ uptake by syncytiotrophoblast. Only DDE (>/=0.70 mug/l) in maternal serum significantly was associated with a small increase in Ca2+ uptake by syncytiotrophoblast. This study will help us determine if low OC contamination significantly modifies Ca2+ transfer in syncytiotrophoblast.

Impact of an enriched-cholesterol diet on enzymatic cholesterol metabolism during rabbit gestation.

An appropriate cholesterol homeostasis is vital for the maintenance and the optimal fetal development. The cholesterol is essential for the synthesis of progesterone and 17beta-estradiol, hormones that actively participate to sustain gestation. However, the administration of 0.2% enriched cholesterol diet (ECD) during rabbit gestation significantly increased the cholesterol blood profile (total-cholesterol, LDL, HDL, esterified-cholesterol and free-cholesterol) of dams and offspring, and induced a reduction of the offspring weight of 15% as compared to the control group. Enzymes involved in cholesterol metabolism (ACAT, HMG-CoA-reductase and cholesterol-7alpha-hydroxylase) are greatly influenced by cholesterol profile. We hypothesized that the administration of an ECD during rabbit gestation modifies the activity of those enzymes. Female rabbits (pregnant or not) were fed with a standard diet or an ECD. At term, livers (dams and offspring) and placentas were collected and ACAT, HMG-CoA-reductase and cholesterol-7alpha-hydroxylase activities were assayed. Our results demonstrate that gestation induced a reduction of ACAT activity (48.9%) in dam's liver and, an augmentation of HMG-CoA-reductase activity (142.4%) whereas it has no effect on cholesterol-7alpha-hydroxylase activity. The administration of the ECD has no additive effect on ACAT, but significantly reduced the HMG-CoA-reductase activity and cholesterol-7alpha-hydroxylase activity as compared with the pregnant control group. In placentas the ECD supplementation has an influence for HMG-CoA-reductase activity, where a 43% increased in observed. Any ACAT activity was detected in placenta and the ECD has no influence on the cholesterol-7alpha-hydroxylase activity. Whereas their offspring's liver present a reduction of ACAT and HMG-CoA-reductase activity. Gestation associated with ECD reduces significantly the HMG-CoA-reductase activity, decreasing the cholesterol synthesis, but placenta seems to compensate this effect by increasing its HMG-CoA-reductase activity.

Expression and role of calcium-ATPase pump and sodium-calcium exchanger in differentiated trophoblasts from human term placenta.

Although placental transfer of maternal calcium (Ca(2+)) is a crucial process for fetal development, the biochemical mechanisms are not completely elucidated. Especially, mechanisms of syncytiotrophoblast Ca(2+) extrusion into fetal circulation remain to be established. In the current study we have investigated the characteristics of Ca(2+) efflux in syncytiotrophoblast-like structure originating from the differentiation of cultured trophoblasts isolated from human term placenta. Time-courses of Ca(2+) uptake by differentiated human trophoblasts displayed rapid initial entry (initial velocity (V(i)) of 8.82 +/- 0.86 nmol/mg protein/min) and subsequent establishment of a plateau. Ca(2+) efflux studies with (45)Ca(2+)-loaded cells also showed rapid decline of cell-associated (45)Ca(2+) with a V(i) of efflux (V(ie)) of 8.90 +/- 0.96 nmol/mg protein/min. Expression of membrane systems responsible for intracellular Ca(2+) extrusion from differentiated human trophoblast were investigated by RT-PCR. Messenger RNAs of four known isoforms of PMCA (PMCA 1-4) were detected. Messenger RNAs of two cloned human NCX isoforms (NCX1 and NCX3) were also revealed. More specifically, both splice variants NCX1.3 and NCX1.4 were amplified by PCR with total RNA of differentiated human trophoblast cells. Ca(2+) flux studies in Na-free incubation medium indicated that NCX played a minimal role in the cell Ca(2+) fluxes. However, erythrosine B (inhibitor of PMCA) time- and dose-dependently increased cell associated (45)Ca(2+) suggesting a principal role of plasma membrane Ca(2+)-ATPase (PMCA) in the intracellular Ca(2+) extrusion of syncytiotrophoblast-like structure originating from the differentiation of cultured trophoblast cells isolated from human term placenta.

Expression of calbindin-D28k (CaBP28k) in trophoblasts from human term placenta.

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.

Calcium fluxes in human trophoblast (BeWo) cells: calcium channels, calcium-ATPase, and sodium-calcium exchanger expression.

Although placental transfer of maternal calcium (Ca(2+)) is a crucial process for fetal development, the biochemical mechanisms are poorly understood. In the current study, we have investigated the characteristics of Ca(2+) fluxes in relation with cell Ca(2+) homeostasis in the human placental trophoblast cell line BeWo. Time-courses of Ca(2+) uptake by BeWo cells displayed rapid initial entry (initial velocity (V(i)) of 3.42 +/- 0.35 nmol/mg protein/min) and subsequent establishment of a plateau. Ca(2+) efflux studies with (45)Ca(2+)-loaded cells also showed rapid declined of cell-associated (45)Ca(2+) with a V(i) of efflux (Ve(i)) of 3.30 +/- 0.08 nmol/mg protein/min. Further identification of membrane gates for Ca(2+) entry in BeWo cells was carried out. Expression of Ca(2+) transporter/channel CaT1 and L-type alpha(1S) subunit was showed by RT-PCR. However, mRNA for CaT2 channel and L-type alpha(1C) and alpha(1D) subunits were not revealed. Membrane systems responsible for intracellular Ca(2+) extrusion from BeWo cells were also investigated. Plasma membrane Ca(2+)-ATPases (PMCA) and Na/Ca exchangers (NCX) were detected by Western blot in BeWo cells. Expression of specific isoforms of PMCA and NCX was further investigated by RT-PCR. Messenger RNAs of four isoforms of PMCA (PMCA 1-4) were detected. The presence of messenger RNAs of two NCX isoforms (NCX1 and NCX3) was observed. Ca(2+) flux studies in Na-free incubation medium indicated that NCX played a minimal role in the cell Ca(2+) fluxes. Inorganic ions such as cadmium and manganese did not modify the Ca(2+) fluxes, however, barium increased cell-associated (45)Ca(2+) by, in part, by reducing radiolabel exit.

Phospholipase C axis is the preferential pathway leading to PKC activation following PTH or PTHrP stimulation in human term placenta.

Parathyroid-related peptide (PTHrP) is abundant in human syncytiotrophoblast where it was suggested to play an important role in maternal-fetal calcium homeostasis. On the other hand, parathyroid hormone (PTH), another hypercalcemic factor, would be implicated in the maintenance of the mother's calcium balance. In many cells, these hormones are associated to G-coupled receptors and activate protein kinase (PKC). Thus, the first aim of this study was to determine the cellular pathway (phospholipase; PLC and phosphatidyl-inositol-3 kinase; PI3K) leading to the activation of PKC following a PTH or PTHrP stimulation in brush border (BBM) and basal plasma membranes (BPM) of human term placenta. Both peptides were shown to be potent modulators of the PKC activity in these membranes with optimal concentrations of 10(-8)M and 10(-9)M for hPTH and hPTHrP, respectively. Furthermore, the use of bisindolylmaleimide (BIM), a non-selective PKC inhibitor, serves to demonstrate the specificity of the PKC-dependent MARCKS-psd phosphorylation. While LY-294002, a PI3K inhibitor failed to counteract the hPTH- and hPTHrP-induced PKC stimulation in BBM and BPM, U-73122, a PLC inhibitor, totally abolished the PKC stimulation by hPTH and hPTHrP. Taken together, these data suggest that the activation of PKC by hPTH or hPTHrP, in BBM and BPM, is preferentially associated to the PLC pathway rather than the PI3K's.

Expression of calcium channels along the differentiation of cultured trophoblast cells from human term placenta.

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 (alpha(1C) and alpha(1D)) 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.

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.

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 Ca(2+)-binding proteins (CaBPs) have an important role in regulating or shuttling cytosolic Ca2+ since they are endowed with a high affinity for Ca2+ (approximately 10(6) M(-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-100alpha, and S-100beta. 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.