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Departments of Human Pathology and Oncology,3
Biomedical Sciences,4
Physiology,5
Molecular Biology,6
Evolutionary Biology,7 University of Siena, 53100 Siena, Italy
Geneva Proteomics Center,8 Central Clinical Chemistry Laboratory, Geneva University Hospital, 1211 Geneva 14, Switzerland
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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calcium, placenta, placental transport, pregnancy, trophoblast
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The growth and development of the human fetus is dependent on the placental transfer of calcium. This mineral is essential for bone formation and for the maintenance of cellular homeostasis and functions. The concentration of calcium in fetal blood greatly exceeds that of maternal circulation, indicating that the mechanism of placenta transfer is an active transport from mother to fetus. Such a process, carried out in vivo by the placental syncytiotrophoblast layer, brings the net increment in calcium accumulation to about 30 g by term, with 80% of the required amount of calcium that enters into the fetal circulation in the third trimester [13]. While providing for the transplacental transport, the syncytiotrophoblasts maintains a total cytoplasmic concentration of calcium estimated to be 1000-fold higher—and a free-calcium concentration several orders of magnitude lower—than that of maternal circulation [14]. This can be achieved by means of cytoplasmic binding processes that regulate the concentration of the free intracellular ion. It is thought that, in the trophoblasts, calcium-binding proteins (CBPs) function in this role [15].
The study reported herein presents data on tissue and cell distribution, expression during pregnancy, and characterization of human placental TPT1. In addition, the effect of TPT1 gene knockdown on the calcium handling of human trophoblast cells has been evaluated. The results are discussed in terms of the potential roles of TPT1 in placental calcium transport.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Lines
The HTR-8/SVneo human trophoblast cell line was kindly provided by Dr. Charles H. Graham (Queen's University, Ontario, Canada). This cell line, established by SV40 Tag immortalization of a short-lived, first-trimester trophoblasts cell line, possesses an extended life span, but it is nontumorigenic and shares phenotypic features with primary cytotrophoblast cell cultures, including expression of cytokeratin, human chorionic gonadotropin, and type-IV collagenase [16]. HTR-8/SVneo cells were maintained in RPMI-1640 medium (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin at 37°C in a humidified atmosphere of 5% CO2.
Tissue Preparation
Placental tissues were obtained from the Tissue Bank of the Hospital of Siena (Siena, Italy). Approval for the study was granted by the local Human Institutional Investigation Committee. An informed consent was obtained from all the patients. First-trimester samples were from patients undergoing elective termination of pregnancy at 6–10 wk of gestation (n = 8). Term placentas (n = 7) were from uncomplicated pregnancy ended by a cesarean section. Placental tissues were immediately rinsed in sterile Hanks balanced salt solution (HBSS) at room temperature to remove excess blood, blotted dry, and carefully dissected with a razor blade. Chorionic villi were extensively washed in sterile HBSS, blotted, and aliquots for protein analysis and RNA extraction were snap frozen and stored in liquid nitrogen. The remainder was fixed in 10% buffered neutral formalin and embedded in paraffin for histology and immunohistochemistry. Sections of each specimen were stained with hematoxylin-eosin and histologically examined by a pathologist. Only tissues from uncomplicated pregnancies were included in the study.
Immunohistochemistry and Immunofluorescence Staining
Immunohistochemistry was performed using the alkaline phosphatase-anti-alkaline phosphatase (APAAP) method [11]. Sections were dewaxed, rehydrated, and washed in Tris-buffered saline (TBS; 20 mM Tris-HCl [pH 7.6], 150 mM NaCl). Antigen retrieval was carried out by incubating sections in sodium citrate buffer (10 mM, pH 6.0) in a microwave oven at 750 W for 5 min. Slides were incubated overnight at 4°C with an anti-human TPT1 monoclonal antibody diluted 1:100 in TBS. Slides were washed and incubated with a rabbit anti-mouse antibody (DAKO, Copenhagen, Denmark) at a dilution 1:30 for 30 min and finally incubated for 30 min with the APAAP complex (DAKO) diluted 1:50 in TBS. The alkaline phosphatase reaction was revealed with new fuchsin and naphthol. Endogenous alkaline phosphatase was blocked by adding 1 mM levamisole to the substrate solution. Sections were mounted and examined under a light microscope. For each case, a negative control was obtained by using the antibody preadsorbed with the recombinant TPT1 at the concentration of 20 µg/ml of diluted antibody. Immunofluorescence staining of TPT1 in trophoblast cells was carried out as described [11]. In brief, HTR-8/SVneo cells were grown on coverslips, fixed in methanol, and incubated with a mouse anti-human TPT1 monoclonal antibody and then with a goat anti-mouse IgG coupled to fluorescein (Cappel, Westchester, PA). Fluorescence microscopy was carried out on a Leica TCS 4-D Laser Scanning Confocal Microscope equipped with a krypton/argon laser (Leica Microsystems, Heidelberg, Germany).
Western Blot Analysis
Frozen placental tissues were thawed, minced with a razor blade, and homogenized on ice three times (20 sec each) with a Polytron (Kinematica, Lucerne, Switzerland) in lysis buffer (50 mM Tris-HCl [pH 7.5], 5 mM magnesium acetate, 0.2 mM EDTA, 0.5 mM dithiothreitol, 10% [vol/vol] glycerol, 0.2% [vol/vol] Triton X-100) supplemented with a protease-inhibitor cocktail containing 4-(2-aminoethyl)benzenesulfonyl fluoride, pepstatinA, E-64, bestatin, leupeptin, and aprotinin (Sigma Chemical Co.). Tissue homogenates were centrifuged at 750 x g for 10 min at 4°C and the supernatant assayed for total protein content [17] and stored at –80°C.
Proteins of first-trimester and term placenta homogenates were fractionated in a polyacrylamide gel in the presence of sodium dodecyl sulfate (SDS) as described [11]. After electrophoresis, the gel was removed and proteins were transferred to nitrocellulose filters (Hybond-C; Amersham Biosciences Corp., Piscataway, NJ) for 1 h on ice in transfer buffer (20 mM Tris [pH 8.3], 190 mM glycine, and 20% methanol). The blot was incubated in blocking solution (BS; 3% dry milk in 10 mM sodium phosphate buffer [pH 7.4], 0.15 M NaCl, 0.1% Triton X-100) for 1 h and then transferred to a 1:2000 solution of an anti-TPT1 monoclonal antibody in BS, incubating overnight at 4°C. Nitrocellulose filters were washed three times with BS and exposed at room temperature for 1 h to an anti-mouse antibody labeled with peroxidase. The blot was washed three times with BS, once with 10 mM sodium phosphate buffer (pH 7.4), 0.15 M NaCl, 0.5% Triton X-100 and twice with 0.05 M Tris-HCl buffer (pH 6.8). For detection, a chemiluminescence kit (Amersham Biosciences Corp.) was used according to the manufacturer's instructions. To normalize for loading differences, after stripping, the blot was exposed to an anti-GAPDH antibody diluted 1:1000 in BS. Densitometric analysis of the bands was carried out using the ImageMaster TotalLab software (Amersham Biosciences Corp.). For TPT1, a negative control was obtained by using the antibody preadsorbed with the recombinant protein at the concentration of 20 µg/ml of diluted antibody.
Reverse Transcriptase-Polymerase Chain Reaction and Quantitative Reverse Transcriptase-Polymerase Chain Reaction
Total RNA was extracted using the SV total RNA extraction kit (Promega Corporation, Madison, WI) following the procedure indicated by the manufacturer. RNA integrity was tested by agarose-gel electrophoresis in the presence of 2.2 M formaldehyde. TPT1 mRNA was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) as described [11]. One microgram of total RNA was diluted in 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 5 mM MgC12 containing 50 U of Moloney murine leukemia virus reverse transcriptase, 20 U of placental RNase inhibitor, deoxy-NTPs (dNTPs; 1 mM each), 2.5 µM oligo d(T) primers (Perkin Elmer) in 20 µl of volume. The mixture was incubated at 42°C for 15 min, 99°C for 5 min, and 5°C for 5 min in a programmable thermal cycler (Bio-Rad). For each RNA specimen, a negative control was prepared by omitting the reverse transcriptase. Gene-specific primers used for PCR were chosen according to the published sequence of the human TPT1 and GAPDH mRNA (GenBank X16064 and NM002046). The TPT1 forward primer was 5'-GAGGGGAAGATGGTCAGTAGG-3', the reverse primer was 5'-TGCTTGATTTGTTCTGCAGC-3', and the expected size of the amplified fragment was 278 base pairs (bp). The GAPDH forward primer was 5'-GAAGGTGAAGGTCGGAGTC-3', the reverse primer was 5'-GAAGATGGTGATGGGATTTC-3', and the expected size of the amplified fragment was 226 bp.
For RT-PCR, 2 µl of RT reaction product were added to a mix containing 5x reaction buffer (300 mM Tris-HCl [pH 8.5], 75 mM (NH4)2SO4, 7.5 mM MgCl2), dNTP mixture (final concentration 0.25 mM), 1.0 U cloned Thermus aquaticus DNA polymerase (Life Technologies), and TPT1 primers (final concentration 0.4 µM) in a volume of 50 µl. PCR was carried out in a programmable thermal cycler (Bio-Rad). Amplifications were carried out for 1 min at 94°C, 1 min at 60°C and 1 min at 72°C for 30 cycles, followed by a final 10 min at 72°C. For each specimen, a blank was prepared using 2 µl of the corresponding RT blank. One fifth of each PCR solution was fractionated by electrophoresis in a 1.8% agarose gel. Gels were stained with ethidium bromide, destained, and photographed. PCR product identity was confirmed by restriction analysis [11]. In brief, the product of TPT1 PCR was extracted with phenol: chloroform and precipitated with ethanol. The amplified fragment was digested with AvaI (Sigma Chemical Co.) following the manufacture's suggested conditions. The products were separated by 2.0% agarose-gel electrophoresis, visualized by ethidium bromide staining, and photographed.
Quantification of mRNA levels in placenta samples was performed by quantitative RT-PCR against standard curves created between 1010 and 100 copies of TPT1 and GAPDH cDNA. Total RNA was retrotranscribed as detailed above and PCR amplification performed using a SYBR Green PCR master mix (Quiagen Inc., Valencia, CA) according to the manufacturer's protocol, on an Opticon 2 Real-Time PCR detection system (MJ Research, Reno, NV). The thermal cycling conditions included 15 min at 95°C and then 40 cycles of amplification for 30 sec at 95°C, 30 sec at 60°C, and 30 sec at 72°C. Melting-curve analysis determined the specificity of the amplified products and the absence of primer-dimer formation. For each experimental sample, the amounts of TPT1 and GAPDH mRNA were determined from the calibration curves. The target amount was then divided by GAPDH to obtain a normalized target value.
Expression and Purification of Recombinant Protein
Recombinant placental TPT1 (rp-TPT1) was expressed in Escherichia coli as described [11]. In brief, PCR was used to synthesize a cDNA encoding the full-length human placental TPT1. Complementary DNA was obtained by reverse-transcription of human placenta RNA.
The PCR product, comprising a 5'-terminal NdeI restriction site and a 3'-terminal BamHI restriction site flanking the cDNA encoding the full length TPT1, was cloned in-frame into the NdeI and BamHI restriction sites of a pET-15b plasmid (Novagen, Madison, WI). The resulting pET-15b/TPT1 expression vector expressed the (His-6)-TPT1 fusion protein. Characteristics of the insert DNA were confirmed by sequencing. The plasmid was used to transform HMS174(D3) E. coli host (Novagen) and the expression of the fusion protein was induced with isopropyl-1-thio-ß-D-galactopyranoside (1 mM). Cells were centrifuged at 4000 x g for 20 min and the pellet resuspended in Tris-HCl 20 mM (pH 7.9), NaCl 500 mM, Igepal 0.1% (vol/vol) and sonicated for 15 sec in ice. After centrifugation at 9000 x g for 20 min at 4°C, the fusion protein was purified to virtual homogeneity by one-step metal affinity on a His-Bind column after elution with a linear gradient of imidazole (0.5 M). Homogeneity of the eluted fractions was evaluated by SDS-PAGE followed by silver staining and characteristics of the fusion protein were confirmed by MALDI-TOF (matrix-assisted laser desorption/ionization-time of flight) mass spectrometry [11].
TPT1 Small Interfering RNA Silencing
TPT1 gene silencing was obtained by transfecting the HTR-8/SVneo cells with a plasmid in which the cloned DNA fragment acts as template for the synthesis of small interfering RNA molecules (siRNA) under the control of the U6 promoter (siStrike U6 Hairpin Cloning System; Promega Corp.). The siRNA targeting TPT1 sequence corresponded to nucleotide 317–337 of human mRNA sequence [7]. As control, a DNA template of the corresponding mouse TPT1 mRNA region, which differs in four nucleotides from the human sequence, was chosen [7]. The DNA sequences constituted the template for generating RNA composed of two sequence motifs separated by a 9-bp spacer to form a double-strand hairpin of siRNA. Oligonucleotides were cloned in the pSiStrike vector, containing a puromicin-resistance gene according to the manufacturer's instructions. The resulting pSiStrike/hTPT1 and pSiStrike/mTPT1 vectors were used to transform JM109-competent cells (Promega Corp.). Recombinant plasmid DNA was purified and used to transfect the HTR-8/SVneo cell line. Cells were grown to 60–80% confluence and then transfected with 2 µg of the pSiStrike/hTPT1 or the control vector (pSiStrike/mTPT1) using a liposome transfection reagent according to the manufacturer's instructions (Lipofectin; Invitrogen). Transfected cells were selected for puromicin resistance for 2 wk. Changes in TPT1 levels were determined by Western blot analysis, as described above.
45Calcium Studies
Calcium binding of rp-TPT1 was evaluated using a 45Ca overlay assay [11]. In brief, 8 µg of the recombinant protein and of control proteins were resolved in a 12% SDS-PAGE and blotted onto nitrocellulose. The membrane was washed in buffer (10 mM imidazole-HCl [pH 6.8], 60 mM KCl, 5 mM MgCl2) and incubated in the same buffer containing 10 µCi/ ml of 45CaCl2 (Perkin-Elmer Life Sciences Inc., Boston, MA) for 20 min. After removing the radioactive solution, the membrane was rinsed twice for 5 min in 30% ethanol and air dried. Protein-associated radioactivity was determined with a PhosphorImager (Amersham Biosciences Corp.).
Ca2+ uptake studies were performed on transfected HTR-8/SVneo cell lines as described by Hershberger and Tuan [18], with minor modifications. Briefly, cells were washed twice with the Ca2+ uptake buffer (Ca/ Mg-free Hanks balanced salt solution; HBSS) and allowed to equilibrate in the same buffer (2 ml) for 15 min. Cells were incubated at 37°C for different intervals of time after the addition of 1 ml of prewarmed (37°C) uptake buffer containing 45CaCl2 (1–2 µCi/well). The incubation was stopped by aspiration of the uptake buffer and by washing three times with ice-cold HBSS. Cells were then solubilized in HBSS containing 1% (vol/vol) of Triton X-100. The cell-associated radioactivity was measured on a ß-counter. The total protein content of each well was evaluated by spectrophotometric quantification, using the bicinchoninic acid (BCA) reagent with BSA as standard. The Ca2+ uptake was expressed as nanomoles of Ca2+ per milligram of protein.
Intracellular calcium levels ([Ca2+]i) were measured on transfected HTR-8/SVneo cells using the Fura-2 method [19]. Cells grown on glass coverslips were loaded for 60 min at 25°C in the dark with 10 µM fura-2 acetoxymethylester (Sigma Chemical Co.). The cells were then washed three times with Ca/Mg-free HBSS and each coverslip placed in a fluorimeter cuvette containing 2 ml of Ca/Mg-free HBSS, at 30°C. Following addition of 1 mM CaCl2, the fluorescence was recorded at the excitation wavelengths of 340 and 380 nm and an emission wavelength of 505 nm in a double-excitation fluorimeter (RF-5000; Shimadzu, Tokyo, Japan). Intracellular Ca2+ levels were calculated from the observed fluorescence ratio 340/380 (R) [19]. In brief, to obtain the minimum fluorescence ratio (Rmin), 400 mM EGTA (pH 8.7) and 16 µM ionomycin were added sequentially, followed by 10 mM CaCl2 to obtain the maximum fluorescence ratio (Rmax) and 5 mM MnCl2 to measure the autofluorescence. After correction of R, Rmin and Rmax for autofluorescence, the [Ca2+]i was determined by the equation
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Statistics
Data analysis was conducted with a statistical software package (GraphPad Prism version 4.02; GraphPad Software Inc. San Diego, CA). The kinetics of calcium uptakes were compared by analysis of covariance (ANCOVA). The Student t-test was used to estimate the levels of significance of differences between the TPT1/GAPDH protein and mRNA ratio of term and first-trimester placenta, as well as the intracellular calcium levels in transfected cells. Values of P < 0.05 were considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Images of sections of human placenta stained with the anti-TPT1 antibody are shown in Figure 1. Intense immunoreactivity was observed in the trophoblast of the chorionic villi of first-trimester specimens, essentially localized in the cytotrophoblast (Fig. 1, A–C). Some of the villous stromal elements were also stained. In term placenta, immunostaining was observed in the villous cytotrophoblast and syncitiotrophoblast (Fig. 1, D–F). Immunoreactivity was also consistently found in extravillous trophoblast cells (not shown). When subcellular localization of TPT1 in the HTR-8/SVneo cells was analyzed by immunofluorescence confocal microscopy, immunoreactive material was mainly found in the cell cytoplasm, with a weaker immunoreactivity visible in the nucleus (not shown).
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Western Blot Analysis
The presence of TPT1 in the placenta was then examined by Western blot analysis. As shown in Figure 2A, the TPT1 antibody recognized a single band of the approximate molecular weight of 22 KDa in the four specimens tested. To gain insight into TPT1 protein-level changes during pregnancy, the assay was repeated on eight first-trimester and seven term placenta specimens. After densitometric analysis, protein levels were expressed as TPT1/GAPDH ratio. As reported in Figure 2B, despite a broad range of individual variation, a statistically significant difference in the protein ratio between first-trimester and term placenta was observed (0.7 ± 0.3 vs. 2.0 ± 0.7 [mean ± SD] P < 0.001).
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RT-PCR
The presence of TPT1 mRNA in human placenta was initially evaluated by RT-PCR analysis. As shown in Figure 3A, an intense band, corresponding in size to the TPT1 product, was obtained from the cDNA of all first-trimester and term placenta specimens examined. The identity of the TPT1 PCR product was then confirmed by restriction analysis. Three randomly chosen 278-bp PCR products were digested with AvaI and yielded two minor fragments of the expected size (202 and 76 bp; Figure 3B). Quantitative RT-PCR analysis of TPT1 mRNA levels carried out on five first-trimester and eight term specimens revealed a statistically significant difference in the TPT1/GAPDH mRNA ratio between first-trimester and term placenta (2.4 ± 0.4 vs. 4.0 ± 1.2 [mean ± SD]; P = 0.017).
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Calcium Studies
To evaluate the calcium-binding activity of placenta TPT1, a 45Ca overlay assay was carried out. The recombinant placental protein, together with equivalent amounts of bovine calreticulin (positive control) and bovine serum albumin (negative control) were exposed to 45Ca2+. As reported in Figure 4, the radioactivity was consistently retained by TPT1 and, as expected, by calreticulin. To examine the relationship between TPT1 levels and the calcium handling in the trophoblast cell, the effect of TPT1 depletion by siRNA gene silencing on calcium uptake and intracellular calcium profile was studied on transfected HTR-8/SVneo cells. Changes in intracellular levels of TPT1 in response to siRNA gene silencing were evaluated by Western blot analysis. Figure 5 shows that TPT1 levels were significantly reduced in the pSiStrike/hTPT1 transfected cells compared with the cells transfected with the control plasmid. The kinetics of cellular calcium uptake of transfected HTR-8/SVneo cells was then examined. As shown in Figure 6, A and B, the kinetic curves of pSiStrike/ hTPT1 transfected cells and control cells had a similar shape, increasing linearly during the first minutes to reach a plateau after 10 min. However, a lower initial velocity (Vi; 11.4 ± 0.725 vs. 18.5 ± 0.8 nmoles of Ca2+/mg protein/min; mean ± SD; n = 6) and a reduced level of cell-associated calcium at plateau (19.1 ± 1.55 vs. 33.8 ± 2.3 nmoles of Ca2+/mg protein; mean ± SD; n = 6) were observed in pSiStrike/hTPT1-transfected compared with control plasmid-transfected cells. When the slopes of the regression lines calculated in the first minutes were compared, statistically significant differences were obtained between pSiStrike/ hTPT1-transfected and control plasmid-transfected cells (10.31 ± 1.46 vs. 17.17 ± 1.56; mean ± SD; F = 10.3, P = 0.018, n = 6). Temporal profile of [Ca2+]i following perfusion with 1 mM of CaCl2 was measured by Fura-2 spectrofluorimetry. As reported in Figure 6C, upon perfusion with CaCl2, an immediate increase in [Ca2+]i was observed in both the transfected cells. However, the rate of increase was significantly higher in pSiStrike/hTPT1-transfected compared with control cells (143.85 ± 54.6 vs. 59.4 ± 18.6 nmoles of Ca2+/min per 1 x 106 cells; mean ± SD; P = 0.026, n = 4).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The transfer of calcium from the mother to the fetus through the placental syncytiotrophoblast results in an accumulation of calcium in the fetal circulation and represents an essential feature of intrauterine calcium metabolism. The initial step of placental calcium transport involves the influx from the maternal blood into the cytosolic compartment of the trophoblast cell, across the placental microvillous membrane. This is followed by calcium translocation through the trophoblast cell cytosol and calcium efflux across the basal plasma membrane into the fetal circulation. Calcium influx proceeds along favorable chemical and electrochemical potentials, likely through Ca2+ channels of the CaT/ ECaC family [20]. On the fetal side, an ATP-dependent Ca2+ transport system is believed to play a main role in the calcium extrusion into the fetal circulation [13]. The process of calcium translocation across the trophoblast cell is less understood. The cytosolic free Ca2+ concentration must be maintained several orders of magnitude lower than extracellular calcium to ensure a constant calcium influx from the maternal plasma [14]. It is believed that a binding system, involving the CBPs of the placenta, might participate in regulating the free cytoplasmic calcium concentration in the trophoblast cell [15]. A number of previous studies focused on the expression of CBPs in the placenta. Among these, the calbindin proteins, a family of cytoplasmic CBPs containing an EF-hand motif, have been extensively investigated. Thus, S100 calcium binding protein G (S100G), calbindin 1 (CALB1), and calbindin-57K (CaBP57K) have been detected in the human and murine trophoblasts [18, 21–23]. Furthermore, an increase in calbindins expression at term, as well as during trophoblastic differentiation, has been reported [18, 21, 23]. These developmental changes have been interpreted as an indication of a role of calbindins in the maternal-fetal calcium transport [18, 21, 23].
The present study showed that TPT1 is a novel CBP of the human placenta and examined the role of the protein in placental calcium transport by downregulating its expression in the trophoblastic HTR-8/SVneo cells and evaluating the effects on intracellular calcium handling. The results point out to an involvement of the protein in placental calcium transport. Three elements are consistent with this hypothesis. First, the tissue and intracellular distribution of TPT1, localized in the trophoblast cell layer and in the cytoplasm of the trophoblast cell. Second, the gestational age-dependent changes in TPT1 expression, resulting in a significant increase of protein and mRNA levels in term placenta. Finally, the alteration of trophoblast calcium uptake and buffering associated with TPT1 gene knockdown.
The gestational changes of TPT1 levels reported in this study raise the question of factors regulating the expression of the protein in the placenta. The effect of hormones, such as parathyroid hormone-related peptide, sex steroids, and vitamin D, on CBPs expression has been previously reported in animal models [15]. Other observations have linked trophoblast differentiation and CBPs expression. Thus, levels of CALB1 [23] and CaBP57K [18] have been shown to increase during differentiation of trophoblast cells. Alteration of CaBP57K levels was also observed to interfere with the process of trophoblast differentiation in vitro [24]. At present, there is no evidence in the literature on hormonal regulation of TPT1 expression. However, experimental data suggested a relationship between cell differentiation and the increase of TPT1 mRNA levels [25]. Current investigations ongoing in our laboratory aim to analyze factors regulating placental TPT1 expression to provide further insight on the role of the protein in human reproduction.
In conclusion, the results of the present study showed the expression of TPT1 in the human placenta and in the trophoblastic cell. In the tissue, the protein was detected in the cytotrophoblast in the first trimester and in the syncytiotrophoblast at term. The expression of the protein significantly increased during gestation, with the higher levels reached in the third trimester. Furthermore, the ability of recombinant human placental TPT1 to bind calcium, as well as the effect of the protein knockdown on intracellular calcium handling, have been demonstrated. Considered together, these results indicate a role for TPT1 in the process of placental calcium transport.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Felice Arcuri, Department of Human Pathology and Oncology, Section of Pathology, University of Siena, Via delle Scotte, 6, 53100 Siena, Italy. FAX: 39 0577 233 235; arcuri{at}unisi.it
Received: 18 March 2005.
First decision: 11 April 2005.
Accepted: 10 June 2005.
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