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Pregnancy |
Laboratoire de Physiologie Materno-foetale,3 Département des Sciences Biologiques, Université du Québec à Montréal, Montreal, Quebec, Canada
Service de pathologie,4 Pavillon St. Luc, CHUM, Montreal, Quebec, Canada
Polypeptide Hormone Laboratory,5 Strathcona Anatomy Building, Montreal, Quebec, Canada
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Calcium, developmental biology, placenta, pregnancy, trophoblast
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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106 M-1] [3] and are believed to function, at least in part, as cytosolic Ca2+ buffers. Several CaBPs, including CaBP9k [4], CaBP57k [5], and S-100 [6] have been localized to the human uterus and placental tissues, but presently CaBP28k has not been identified either in human trophoblasts [7] or in rat trophoblasts [8], although it has been detected in the mouse placenta [9]. In addition, Müller et al. [10] described the presence of CaBP28k in human placenta while evaluating the expression of various human tissues for the presence of CaBP28k by PCR, but a confirmation of the findings by sequencing remains elusive. CaBP28k was the first known target of vitamin-D action and has been initially identified in the avian intestine, where it occurred in high concentrations [3]. Since then, it has been reported in many other tissues such as mammalian kidney, brain, islets of the pancreas [11], and bone [11, 12]. CaBP28k shares 58% homology with calretinin [13], but despite their similarities in size and sequences, they are localized to distinct subsets of cells such as brain cells and seem to have different functions. The contrary is true for CaBP9k and CaBP28k. Although there is no direct affiliation between the CaBP9k and CaBP28k genes, and there are no similarities between the sequences of the two proteins [12, 14, 15], both have been identified in the placenta in mice. Consequently, the wide distribution of CaBP28k among species and tissues and its evolutionary conservation suggests that it plays a fundamental metabolic role [12]; however, the precise nature of this role is still a matter of debate. The affinity of CaBP28k for Ca2+, together with its high concentration in certain tissues, has led to the speculation that it acts as an intracellular Ca2+ buffer, carrier, or enzyme modulator [12]. In the placenta, a suitable buffering system could involve one or more of the several placental CaBPs (including CaBP28k) because, when translocating across the cell, the trophoblast must maintain free Ca2+ concentrations in its cytoplasm that are several orders of magnitude lower than in the extracellular space [16].
The human placental trophoblasts carrying out nutrient transport are located mainly in the placental villi and chorionic villi facing the maternal blood [17], where they appropriately mediate active Ca2+ transport to the fetus [18]. Thus, human villous cytotrophoblast cells form syncytial trophoblasts through a process of cell fusion [19] and time-dependent biochemical and morphological differentiation [20]. At the end of pregnancy, the placental syncytiotrophoblast becomes a continuous epithelial layer by terminal differentiation of the underlying cytotrophoblast cells covering the entire maternal surface of the human placenta [21, 22], forming barriers between the mother and fetus across which the required nutrients of the fetus must be translocated. In addition, the syncytiotrophoblasts perform numerous specialized functions, including mediating the transport immunoglobulins from the maternal to fetal circulation and functioning as an endocrine organ, secreting hormones such as hCG [23].
The aim of this study was to unequivocally demonstrate for the first time that CaBP8k is present in placental tissues and in vitro cultured trophoblasts from two different placental development stages of the human term pregnancy. Furthermore, we wish to establish whether there is any correlation between the increase in CaBP28k expression in differentiated cells compared with undifferentiated cells and the Ca2+ uptake together with hCG secretion in the placenta.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Human embryonic kidney-293 (HEK-293) cells (ATCC No. CRL1573, American Type Culture Collection, Manassas, VA) were used as a positive control because they have been shown in the kidney. Indeed, the energy-dependent component of Ca2+ reabsorption involves a cytosolic transporting protein, CaBP28k, which is the main renal protein [12]. These cells were grown at 37°C under 5% CO2 in air atmosphere in Dulbecco modified eagle medium (DMEM) (Life Technologies Inc., Burlington, ON, Canada), supplemented with 10% fetal bovine serum and 1 x penicillin-neomycin-streptomycin mixture (1 x PNS) from a 100 x PNS stock (GibcoBRL, Burlington, ON, Canada). For experiments, exponentially growing HEK-293 cells were dispersed with trypsin-EDTA solution (GibcoBRL) and replated on coverslips in 58- x 115-mm culture dishes for 72 h. Cells were then fixed in 6% paraformaldehyde before immunochemical detection.
Cytotrophoblast Cells Isolation and Culture
Human term placentas from 37 to 40 wk gestation were obtained immediately after spontaneous vaginal delivery, in accordance with the established guidelines of the ethical committee of St. Luc Hospital and the University of Quebec in Montreal, QC, Canada. The cytotrophoblasts were isolated from a 5% to 75% discontinuous Percoll gradient according to the method of Kliman [13]. The isolated trophoblasts were resuspended in DMEM containing 10% fetal calf serum containing 1 x PNS. The cells were plated at a density of 1.6 x 106/well on coverslips in 24-well plates (Sarstedt, Montreal, QC, Canada), gassed with 5% CO2 in air at 37°C. The culture medium was subsequently changed every 24 h for a maximum of 96 h.
In vitro expression of CaBP28k was assessed at 2 and 96 h using plated trophoblasts incubated at 37°C in 5% CO2 atmosphere. Trophoblasts were fixed in 6% paraformaldehyde.
Purity of Cytotrophoblast Cells
The purity of freshly isolated cytotrophoblast cells was assessed by measuring cytokeratin 7 binding to cell-specific antigens by flow cytometry. Briefly, cytotrophoblast cells were resuspended in PBS (120 mM NaCl, 2.7 mM KCl, 10 mM phosphate, pH 7.4) (Sigma, St. Louis, MO), centrifuged, then fixed in methanol at -20°C for 20 min, After extensive washes in PBS, cells were incubated in BSA (1:50) for 30 min to block any nonspecific binding. Cells were then incubated in the presence of a FITC monoclonal mouse anticytokeratin peptide 7 (Axyll, Westbury, CT), a cytotrophoblast cells–specific antibody diluted at 1:600 in PBS containing 1% BSA for 45 min at room temperature in the dark. Controls were performed by using mouse isotype IgG. After extensive washes, cells were resuspended in PBS. Flow cytometry data were acquired using a FACScan system (Becton Dickinson, San Jose, CA) equipped with an Argon-ion (488 nm) laser. Dead cells and debris were excluded from the analysis. Five thousand cells were analyzed with CellQuest software (Becton Dickinson). Cytotrophoblast cells purity was measured as the percentage of total cells positive for cytokeratin peptide 7 (cytokeratin 7). Only cells containing >95% purity were used in this work.
Hematoxylin Staining
At Day 4, syncytiotrophoblasts grown on coverslips were stained for a few minutes with Gaot's hematoxylin made of an equal volume of 2 g of NH3 Fe(SO4)2 and 2.4 ml of H2SO4 mixed with 1.5 g of hematoxylin dissolved in 95% ethanol. After extensive washes with tap water, cells were mounted on slides and then examined by light microscopy at 60x magnification.
Human CG Measurement
Trophoblast hCG levels in conditioned media were determined using a commercially available hCG ELISA kit (Medicorp, Montreal, QC, Canada). Conditioned media were harvested daily from trophoblast cultures, centrifuged, and frozen at -20°C. Samples were applied in a 96-well dish coated with an antibody against hCG. A second antibody against hCG conjugated to horseradish peroxidase (HRP) was added to each well and, subsequently, the antigen-antibody complex was revealed by 3,3',5,5'-tetramethylbenzidine, a chromogenic substrate for HRP.
45Calcium Uptake Studies
Ca2+ uptake studies were performed on trophoblasts after 1 and 4 days of culture. Briefly, cells were washed twice with the Ca2+ uptake buffer (Hanks balanced salt solution containing 1.26 mM CaCl2, 10 mM HEPES, and 0.1% BSA) and allowed to equilibrate in the same buffer (250 µl) for 10 min. Thereafter, cells were incubated at 37°C for different intervals of time after the addition of 250 µl of uptake buffer containing 45CaCl2 (2–4 µCi/well,), for which specific activity is 8–25 mCi. The incubation was stopped by aspiration of the uptake buffer. The cells were washed three times with 1 ml of ice-cold PBS containing 4 mM EGTA (to eliminate the nonspecific component of the uptake), then solubilized in 0.1 M NaOH. The cell-associated radioactivity was measured by a ß-scintillation 1400 Wallace counter. The cellular protein content of each well was evaluated by spectrophotometric quantification, using the bicinchoninic acid (BCA) reagent with BSA as standard. The Ca2+ uptake is expressed as nmole of Ca2+ (from specific activity) per milligram of cellular proteins.
RNA Isolation and Northern Blot Analysis
Total RNA was isolated from cytotrophoblast cells, syncytiotrophoblasts, and HEK-293 cells, using TRIzol (GibcoBRL) followed by chloroform extraction and isopropanol precipitation. Thirty micrograms of total RNA were then subjected to denaturating formaldehyde-agarose (1% w/v) gel electrophoresis, transferred to hybond-N nylon membrane, and UV cross-linked. The oligonucleotide 5'-CTC TTC TGT GGG TAA TAC GTG AGC CAA CTC - 3' corresponding to the antisense sequence of the CaBP28k was used as a specific probe and 5'-end labeled using the T4 polynucleotide kinase (Roche Diagnostics, Laval, QC, Canada) and 
32P [ATP]. The membrane was prehybridized for 5 h at 50°C in hybridization buffer (1 M NaCl, 100 mM Tris pH 7.4, 6 mM EDTA, 2 x Denhardt's [0.2 Ficoll, 0.2 g polyvinylpyrrolidone, and 0.2 g bovine serum albumin in 500 ml distilled water], 0.02% SDS, 100 µg/ml DNA salmon sperm) and then hybridized for 16 h at 50°C in the same solution containing 107 cpm/ml of the labeled oligonucleotide. After three washes of 15 min at room temperature in 2 x SSC (0.15 NaCl, 0.015 M sodium citrate, pH 7.0)-0.1% SDS, the membrane was exposed overnight at - 80°C to Kodak XOMAT films (Eastman Kodak, Rochester, NY). For correction of differences in mRNA loading, the membrane was subsequently hybridized with an 18S cDNA probe labeled with [
32P]deoxy-CTP using the T7 QuickPrime kit (Pharmacia Biotech, Mississauga, ON, Canada). Densitometric quantification of the signals was then performed using a BioRad densitometer (Model GS-700, BioRad Laboratories, Hercules, CA).
Complementary DNA Synthesis, RT-PCR, and Sequencing
A 200-µg aliquot of total RNA was used to isolate mRNA, using a Qiagen kit as per manufacturer's instructions (Qiagen, Mississauga, ON, Canada). Complementary DNAs were prepared from human placental trophoblasts and HEK-293 cells mRNA, using the Omniscript reverse transcriptase kit (Qiagen), 10 µM hexamere final concentration, and 0.5 U/µl RNase inhibitor (Amersham Pharmacia Biotech, Baie d'Urfé, QC, Canada) in a total of 20 µl total reaction at 37°C for 1 h.
PCR products were amplified by using 5 µl of the resulting cDNA. Specific primer pairs (Operon, Seattle, WA) were chosen according to the published sequence of the human CaBP28k mRNA (GenBank, accession no. NM004929). The forward primer was located at nucleotides 198–218 of human mRNA CaBP28k, and the reverse primer was positioned at nucleotides 923–946. The following protocol was used in Perkin Elmer 24000 thermal cycler (Perkin Elmer, Wellesley, MA): 94°C for 4 min hot start followed by 94°C for 1 min, 60°C for 1 min, 72°C for 1 min for 34 cycles then 72°C for 7 min. A negative control without any cDNA was also included in each PCR.
The integrity of the cDNAs from the trophoblast cells and HEK-293 cells were checked by the presence of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (accession no. XM006959) housekeeping gene detected by PCR, using specific primers (Operon) as follows: forward primer (5'-GAG TCA AC GAT TTG GTC GTA TTG-3') and reverse primer (5'-GCT GTA GCC AAA TTC GTT GTC-3'). GAPDH was amplified for 25 cycles.
All PCR products were visualized on an agarose gel with ethidium bromide staining, using a DNA molecular mass marker of 100 base pairs (bp) (GibcoBRL). Relative amount of CaBP28k products were estimated by digital densitometry with quantitative software (Alpha Innotech Corporation, San Leandro, CA). CaBP28k mRNA expression were normalized relative to the expression of GAPDH mRNA. The analyses of PCR products were carried out in three separate experiments.
All RT-PCR products were sequenced at the Operon Qiagen Facility (Seattle, WA) to confirm previous results. Mapping of cDNA fragment obtained was determined at The National Center for Biotechnology Information Web site (http://www.ncbi.nlm.nih.gov/).
Tissue Sections and Immunochemistry
The distribution of CaBP28k in trophoblast tissues of the human placenta was assessed using five fresh placenta sections from each of three different placentas, which were rapidly frozen in cold methylene butane and immersed in Tissue-Tek O.C.T. cryo-embedding medium (Miles Labs., Elkart, IL). Blocks were sectioned at 5 µm with a cryomicrotome. Sections were then fixed in 6% paraformaldehyde for 5 min.
Immunohistochemical and immunocytochemical analyses of CaBP28k were performed with a monoclonal anti-CaBP28k Mouse IgG1 antibody (mAb) (Sigma). Any nonspecific binding on fixed frozen sections mounted on slides was blocked with 2% BSA in 1% PBS-Tween for 3 h at room temperature. After permeabilization in 0.03% saponin, the sections were exposed to the primary antibody (1:400) for 1 h. After three 10-min washes with PBS in 0.05% Tween 20, sections were exposed to Alexa 488-labeled goat anti-mouse IgG (Molecular Probes, Eugene, OR) (1:6000) for at least 30 min at room temperature. The expression of CaBP28k by HEK-293 cell line, cytotrophoblast cells, and syncytiotrophoblasts was revealed on different coverslips by using the same conditions and the same antibody as for the tissue sections. Replacing primary antibody with an equal concentration of nonimmune mouse serum assessed specificity of immunolabeling for placental tissues, HEK-293 cells, and trophoblasts. For nuclear DNA labeling, 500 µg/ml RNase A (Sigma) was then added after secondary antibody for 30 min followed by 2 µg/ ml propidium iodide (Molecular Probes).
All observations were performed at 60x magnification on tissue sections and monolayer cells labeled for each slide and coverslips by using a Nikon Eclipse TE 300 (Nikon, Tokyo, Japan) equipped with a confocal laser-scanning microscope (BioRad MRC1024).
CaBP28k Protein Precipitation and Western Blot Analysis
Cytosolic fractions from cytotrophoblast cells, syncytiotrophoblasts, and HEK-293 cells were prepared by spinning lysis homogenate at 10 000 g for 20 min at 4°C as described by Dutta-Roy et al [24]. The resulting supernatants were stored at -80°C until further use. Protein concentration was determined by Pierce BCA assay (Pierce, Rockford, IL). Five hundred microliters of cytosolic CaBP28k (600 µg) was precipitated with an equal volume of 20% trichloroacetic acid for 30 min at 4°C, and 500 µl of cold acetone was added to the pellet. After centrifugation, pellets were dissolved in Lammeali buffer, fractionated by Tris-glycine SDS-polyacrylamide gel electrophoresis, then electrotransferred onto a polyvinylidene difluoride (PVDF) membrane for 1 h. Any nonspecific binding was blocked with 5% BSA in 1% Triton X-100 Tris-buffered saline (TBS-T) for 60 min at room temperature. The PVDF membrane was then probed for the presence of CaBP28k by incubation with primary antibody mouse anti-CaBP28k antibody (Sigma) (diluted 1:400) for at least 1 h. After washing in TBS-T, the PVDF membrane was incubated with HRP–anti-mouse IgG fraction of goat monoclonal antiserum secondary antibody (Chemicon International, Temecula, CA) diluted 1:10 000. Finally, the CaBP28k protein was revealed by using a chemiluminescence Western blotting plus kit (NEN Life Science Products, Boston, MA). The negative control for nonspecificity was done on the PVDF membrane by using nonimmune mouse IgG in place of the first antibody. The PhosphorImager software 2200 was used to quantify the changes in intensity of various bands. The Western blot analyses were carried out on three separate experiments.
Data Analysis
Statistical analyses were performed using a nonlinear regression model test for the kinetic of Ca2+ uptakes, and the means of hCG secretion were compared using an approximate Student t-test with unequal variances and positively correlated samples. The results were presented as the mean ± SEM and were considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Purity of the freshly isolated cytotrophoblast cells was assessed by flow cytometry and expressed as percentage of total positive cells for cytokeratin 7. As shown in Figure 1A, only cell isolates containing >95% cytotrophoblast cells were used for this study. Cytotrophoblast cells controls scarcely expressed cytokeratin 7 (Fig. 1B), ruling out the possibility of any contamination by syncytial tissues.
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Light microscopy examination of cell cultures showed that, within 4 days after plating, the cultured cytotrophoblast cells formed cell aggregates consisting mostly (99%) of large areas containing multiple dark blue nuclei corresponding to functional syncytiotrophoblasts (Fig. 2). Quantitative counts of nuclei revealed at least 90% of the nuclei were found in syncytial cells. There was no appreciative nuclear replication as evidenced by the absence of mitotic figures. At this time of culture, no single mononuclear cells were observed. Cells seem to form a network that corresponds to functional syncytiotrophoblasts in terms of their ability to secrete hCG.
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To assess whether the syncytiotrophoblasts formed in vitro were functional, we examined cultures between Day 1 and Day 4 for the expression of hCG in the culture media by using an hCG-ELISA kit. Figure 3 shows the data of seven experiments on the temporal pattern of hCG release from cultured trophoblasts. The mean of hCG secretion in the culture media was negligible during the first day of plating when compared with Day 2 (P < 0.001). The mean increases drastically from Day 2 to Day 3 (P < 0.001); however, this raise was less significant (P < 0.1) when the mean secretion of Day 3 and Day 4 were compared. Consequently, the absolutes values of hCG release in the media were variable in day of culture, with the highest level of secretion at Day 4 of culture for all the placentas tested.
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45Ca Uptake by the Trophoblasts
Ca2+ uptake experiments on trophoblasts were performed at Day 1 and Day 4 of culture. The kinetics of Ca2+ uptakes for Day 1 were rapid for the first 2 min of incubation with an initial velocity (Vi) of 1.946 ± 0.077, followed by the establishment of a plateau of 6.8 ± 0.74 nmol/mg protein at 30 min incubation (Fig. 4). The kinetic of Ca2+ uptake at Day 4 of the cultured trophoblasts has a similar shape as compared with Day 1, with a higher Vi of 4.673 ± 0.28 (2.45-fold compared with Day 1) and a plateau of 14.03 ± 1.509 (2.06-fold compared with Day 1). The Vi of Ca2+ uptake, along with the time of culture, correlated with the relation observed between days of culture and the plateau. In addition, the nonlinear regression determination coefficients (R2) of the Ca2+ uptake for Day 1 and Day 4 were 85% and 79%, respectively, indicating a good fit of the model to the data.
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Detection of a CaBP28k Transcript in Trophoblasts
The expression of a CaBP28k transcript in cytotrophoblast cells, syncytiotrophoblasts, and HEK-293 cells was measured by Northern blot analysis using a synthetic 30-bp oligonucleotide as a probe (see Materials and Methods). A single transcript of 2.58 kb was detected according to the sequence of GenBank in all the cell types tested (Fig. 5A), with the highest expression in syncytiotrophoblasts (2.5-fold) followed by HEK-293 cells (1.7-fold) and cytotrophoblast cells (1.02-fold) after normalization with 18S expression levels (Fig. 5B).
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To further confirm these results, cDNAs generated from cytotrophoblast cells and syncytiotrophoblasts were evaluated for the presence of CaBP28k transcript by RT-PCR analysis as shown in Figure 6A. Using primers specific for human CaBP28k, RT-PCR undeniably revealed the presence of a CaBP28k single transcript of 748-bp product (Fig. 6A) corresponding to the expected size amplified in both cell types with a greater abundance (3.52-fold) in syncytiotrophoblasts grown for 4 days at 37°C, and HEK-293 cells expressed 1.80-fold compared with freshly isolated cytotrophoblast cells (1.26-fold) after normalization with GAPDH expression levels. The integrity of cDNA samples was confirmed by the presence of a 945-bp PCR product corresponding to the GAPDH housekeeping gene (Fig. 6B).
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The CaBP28k fragments obtained by RT-PCR were sequenced. Mapping of cDNA fragments was determined at The National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/), and the fragments match the CaBP28k mRNA corresponding fragment.
Expression of CaBP28k Placenta Sections and In Vitro Cultures
To investigate whether CaBP28k was expressed by trophoblasts of a normal full-term placenta, immunohistochemical staining of paraformaldehyde fixed tissue sections was performed using anti-CaBP28k mAb. Figure 7A shows that CaBP28k is expressed in syncytiotrophoblast as well as cytotrophoblastic tissues. The staining seemed to be mainly cytoplasmic and were similar between the different sections of the placenta. No staining was observed in the section stained with nonimmune mouse serum, indicating the specificity of the reaction (Fig. 7B).
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In vitro cultures of HEK-293 cells, cytotrophoblast cells, and syncytiotrophoblasts stained with anti-CaBP28k revealed staining in HEK-293 cells (Fig. 8A), undifferentiated cells (Fig. 8B), and differentiated cells (Fig. 8C), with an overall higher level in syncytiotrophoblasts. All the controls failed to express any positive staining when cells were incubated only with nonimmune serum (Fig. 8A'–C').
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Western Blot Analysis
By using mouse anti-CaBP28k, a single band was observed from cytotrophoblast cells, syncytiotrophoblasts, and HEK-293 cells, indicating that the antibody is specific for a 28-kDa protein (Fig. 9). No bands were visible in the PVDF membranes used for negative control (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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This study describes for the first time the consistent distribution of in vivo– and in vitro–expressed CaBP28k in human placental cytotrophoblast cells and syncytiotrophoblasts isolated from different individuals. These two types of cells represent two physiological stages of differentiation of human trophoblasts that are intimately involved in the transport of nutrients from the mother to the fetus by the placenta. In vitro culture of cytotrophoblast cells showed that, from Day 1, the freshly isolated cytotrophoblast cells consistently transform into aggregates after spontaneous migration toward each other, and syncytia eventually develop through fusion [19]. Simultaneous with this transformation, the cytotrophoblast cells acquire specific endocrine functions, including secretion of hCG [19], confirming previous reports [19, 31]. Thus, during the first day, the hCG secretion was very modest, suggesting that our isolated cytotrophoblast cells were very highly purified and free of any syncitial fragments. This finding was confirmed by the presence of more than 95% cytokeratin 7 positive cells, only expressed by cytotrophoblast cells [32].
It is noteworthy that Ca2+ uptake profile correlates exactly with the hCG secretion pattern, which increases with differentiation. Indeed, Ca2+ uptake increased during the differentiation period and was very high at Day 4. Ca2+ transport mechanisms in human trophoblasts include carriers such as CaBPs. The presence of both CaBP9k and CaBP28k in mouse placenta [9] suggests that they may be involved in transcellular Ca2+ transport. Hence, in the enterocyte for example, the Ca2+ is immediately bound to cytosolic CaBP9k and transferred across the cell by facilitated diffusion. The binding of Ca2+ to CaBP9k maintains the intercellular free (ionic) Ca2+ concentration below the 10-7M necessary for cellular homeostasis. The CaBP9k-bound Ca2+ is then transferred to Ca2+-ATPase for export from the cell [33]. Clearly, this system could be applied to the placental CaBP28k. Indeed, considering both Vi and plateau, we observed that Ca2+ uptakes during Day 1 were significantly low when compared with Day 4, which suggested that the cytotrophoblast cells had a lower capacity to transport Ca2+ compared with syncytiotrophoblasts. In addition, CaBP28k expression, which was higher at Day 4 of cell cultures compared with Day 1, may contribute to the enhanced Ca2+ uptake. It also seems that the increase of Ca2+ transport during differentiation may stimulate CaBP28k mRNA and gene expression levels and probably protein production. Indeed, Northern blot analysis showed a transcript of 2.5 kb highly expressed in syncytiotrophoblasts when compared with cytotrophoblast cells. These results were confirmed by RT-PCR, which revealed a presence of a 748-bp CaBP28k transcript in the different cell types tested with a significantly greater expression (3.52-fold) in syncytiotrophoblasts or HEK-293 cells (1.80-fold) compared with freshly isolated cytotrophoblast cells (1.26-fold) after normalization with GAPDH expression levels. In addition, careful examination of immunochemically stained cytotrophoblast cells and syncytiotrophoblasts showed that the anti-CaBP28k labeling was associated with both types of cells in placental tissues as well as in cultured cells. In our cultured cells, immunostaining was mainly located in the cytoplasm, which may indicate that transcellular Ca2+ transport may be based on facilitated diffusion such as in enterocytes. However, the mechanisms by which CaBP28k increases Ca2+ transport remain unclear [34].
There is at least one explanation for the effect of increased CaBP28k levels on Ca2+ uptake, i.e., the higher level of CaBP28k could enhance the buffering capacity of cells or stimulate the Ca2+ entry mechanism. In this way, Hussain and Mughal [35] suggested that CaBP28k might act as an intracellular shuttle to facilitate the transport of Ca2+ through the trophoblastic cytosol. Regarding kidneys, Bouthiauy et al. [34] hypothesized that CaBP28k may increase Ca2+ uptake by binding the transported Ca2+, thus increasing the ionized gradient from the tubular lumen to the microenvironment at the internal surface of the membrane, maintaining this gradient despite the constant influx of Ca2+. In our work, differentiated cells expressing a high level of CaBP28k exhibited significantly higher cellular Ca2+ uptake activity compared with undifferentiated cells. Therefore, at the entry of the cells, CaBP28k may try to bind and retain as much as Ca2+, thus explaining the higher plateau in syncytiotrophoblasts. Furthermore, CaBP28k may play a direct role in Ca2+ storage when trapped within certain organs such as the bones of the growing fetus. Apparently, CaBP28k knockout mice may provide direct evidence that CaBP28k acts as a Ca2+ buffer as an important modulator of induced intracellular Ca2+ transients [36]. Interestingly, in the chicken shell gland, an analogous organ to the uterus, an active Ca2+-ATPase is localized on the gland cell apex [33], where it pumps Ca2+ out of the gland in the presence of uniformly distributed CaBP28k [37].
In conclusion, the observation that CaBP28k transcript is transiently less detectable in undifferentiated cells such as cytotrophoblast cells suggests that it may be involved in Ca2+ transport or cell development in human trophoblast through Ca2+ buffering. Because the exact protein turnover rate was not determined in the present study, it is difficult to identify the most likely scenario. Moreover, CaBP28k expression might be under the control of other factors involved in Ca2+ homeostasis and thus liable to an upregulation to handle the increasing Ca2+ requirements of the developing fetus. Ongoing investigations aim to further analyze the exact mechanisms of Ca2+ uptake in correlation with the modulation of CaBP28k expression. Under several experimental conditions, the mRNA turnover will be measured as well as the regulation of the CaBP28k gene transcription using cell lines transfected with the CaBP28k promoter linked to a reporter gene.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Julie Lafond, Laboratoire de Physiologie Materno-f
tale, Département des Sciences Biologiques, Université du Québec à Montréal, C.P. 8888, Succursale "Centre-Ville", Montréal, QC, Canada H3C 3P8. FAX: 514 987 4647; lafond.julie{at}uqam.ca
Received: 15 July 2002.
First decision: 1 August 2002.
Accepted: 19 December 2002.
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