Expression and regulation of 4F2hc and hLAT1 in human trophoblasts

Yoko Okamoto, Masahiro Sakata, Kazuhiro Ogura, Toshiya Yamamoto, Masaaki Yamaguchi, Keiichi Tasaka, Hirohisa Kurachi, Masato Tsurudome, and Yuji Murata

Department of Obstetrics and Gynecology, Osaka University Faculty of Medicine, Osaka 565-0871; and Department of Microbiology, Mie University School of Medicine, Tsu, Japan


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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The neutral amino acid transport system L is a sodium-independent transport system in human placenta and choriocarcinoma cells. Recently, it was found that the heterodimer composed of hLAT1 (a light-chain protein) and 4F2 heavy chain (4F2hc), a type II transmembrane glycoprotein, is responsible for system L amino acid transport. We found that the mRNAs of 4F2hc and hLAT1 were expressed in the human placenta and a human choriocarcinoma cell line. The levels of the 4F2hc and hLAT1 proteins in the human placenta increased at full term compared with those at midtrimester. Immunohistochemical data showed that these proteins were localized mainly in the placental apical membrane. Data from leucine uptake experiments, Northern blot analysis, and immunoblot analysis showed that this transport system was partially regulated by protein kinase C and calcium ionophore in the human choriocarcinoma cell line. Our results suggest that the heterodimer of 4F2hc and hLAT1 may play an important role in placental amino acid transport system L.

amino acid transport system L; protein kinase C; calcium ionophore


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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FACILITATED DIFFUSION and active transport are responsible for transport of substances such as glucose and amino acids, respectively. Glucose is transported by sodium-independent facilitated diffusion along a concentration gradient (4). The mechanism for placental glucose transport depends on the cellular membrane carrier proteins facilitative glucose transporters 1 and 3 (GLUT-1 and -3), which have been the subject of several reports (2, 5, 36, 41). On the other hand, active placental amino acid transport is thought to be mediated by specific carrier proteins, which have not been fully characterized.

On the basis of the nomenclature developed originally by Christensen et al. (10), amino acid transport systems such as L, y+L, A, and others have been identified in the placental syncytiotrophoblasts by means of a filtration method employing membrane vesicles (15, 17). Among these systems, amino acid transport system L in the human placenta and human choriocarcinoma cells is a sodium-independent transport system that is noted for its preferential affinity for hydrophobic neutral amino acids, including branched-chain and aromatic amino acids (17, 34). However, only a few studies of the regulation of the activity of amino acid transport system L have been reported (7, 34). Measurement of the uptake of the radiolabeled amino acids into cells revealed that system L transport activity is affected by protein kinase C and extracellular pH in the JAR human placental choriocarcinoma cell line (7, 34). Recently reported evidence also indicates that growth factors such as insulin, insulin-like growth factor I, and epidermal growth factor regulate other systems of placental amino acid transport (6, 19). However, more detailed studies involving different cell types, protein kinases, growth factors, and other effectors are needed to understand the regulation of the activities of system L and other amino acid transport systems.

The 4F2 (CD98) cell surface antigen is a 120-kDa disulfide-linked heterodimer composed of an ~80-kDa heavy chain and an ~40-kDa light chain (13, 14). The 4F2 antigen was originally identified as a lymphocyte activation antigen (13, 14), but its function has not been clarified. Recently, various members of a novel family of glycoprotein-associated amino acid transporters have been identified and shown to play an important role in cellular uptake and/or basolateral extrusion of basic and neutral amino acids. These permease-related proteins contain 12 putative transmembrane domains and require heterodimerization with a type II transmembrane heavy-chain protein such as 4F2hc to express their function as transporters. LAT1 (18, 23, 24, 26), y+LAT1 (31, 42), y+LAT2 (31, 42), xCT (38), and LAT2 (32, 40) have been shown to be linked with 4F2hc. The heterodimer composed of LAT1 and 4F2hc is known to be responsible for sodium-independent neutral amino acid transport (L-type transport) (18, 23, 24, 26). LAT2 also mediates system L transport with 4F2hc; however, LAT2 transports large and small neutral amino acids with lower affinity than LAT1 (32, 40). Other proteins such as y+LAT1 and y+LAT2 are associated with y+L-type transport, which transfers basic amino acids in a sodium-independent manner and neutral amino acids in a sodium-dependent manner (31, 42), while the xCT-4F2hc heterodimer transports cystine/glutamate (38). Progress in the molecular biological analysis of amino acid transport systems has thus enabled us to characterize the expression and regulation of the system L transport components hLAT1 and 4F2hc in human placenta and choriocarcinoma cells.

In this study, we examined whether 4F2hc and hLAT1 mRNAs are expressed in the human placenta and a human choriocarcinoma cell line. Next, we studied the gestation-dependent expression and localization of 4F2hc and hLAT1 in the human placenta. Finally, we examined the regulation of system L amino acid transport by protein kinase C and a calcium ionophore in the choriocarcinoma cell line.


    MATERIALS AND METHODS
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MATERIALS AND METHODS
RESULTS
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Chemicals. 4beta -Phorbol 12-myristate 13-acetate (PMA), A-23187 (calcium ionophore), EDTA, aprotinin, leupeptin, phenylmethylsulfonyl fluoride, and 2-amino-2-norbornane-carboxylic acid (BCH) were obtained from Sigma (St. Louis, MO). L-[4,5-3H]leucine (69.0 Ci/mmol) and a multiprime DNA labeling kit were purchased from Amersham (Arlington Heights, IL). [alpha -32P]ATP (3,000 Ci/mmol) was purchased from ICN Biomedicals (Irvine, CA).

Cell culture. BeWo cells, human mononuclear cytotrophoblast-like choriocarcinoma cells, were obtained from the Health Science Research Resources Bank (Osaka, Japan). Cells were maintained in culture medium (Ham's F-12K with 0.12% NaHCO3, pH 7.4, 50 mg/l streptomycin, and 50 kU/l penicillin G) containing 15% fetal bovine serum in an atmosphere of 95% air-5% CO2 at 37°C. Confluent cells were treated with 0.02% EDTA and 0.25% trypsin, and the trypsinized cells were transferred to new flasks at a density of 1 × 104/cm2. The trypsinized cells were plated in 24-well disposable Falcon dishes at a density of 1.0 × 105 cells/well and allowed to grow as a monolayer until confluency.

Measurement of leucine uptake. Uptake of L-[4,5-3H]leucine was measured in BeWo cells plated in 24-well plates as described previously (6 wells per group) (37). The medium was removed from the monolayer cultures, and each well was washed rapidly twice with a buffer containing 25 mmol/l HEPES, pH 7.5, 140 mmol/l NaCl (or 140 mmol/l choline chloride instead of NaCl), 5.4 mmol/l KCl, 1.8 mmol/l CaCl2, and 0.8 mmol/l MgSO4 (uptake buffer). After the cells had been washed, leucine uptake was measured by addition of 1 ml of uptake buffer containing 10 nmol/l radiolabeled leucine. BCH (1 mmol/l) was used for the inhibition of L system-mediated uptake. After 5 min of incubation, the radioactive buffer was removed, and each well was rapidly washed twice with the uptake buffer. The cells were solubilized in 1 ml of 0.03% sodium dodecyl sulfate (SDS), the lysate was transferred to a scintillation vial, and the radioactivity of the contents was measured with a liquid scintillation counter.

Leucine uptake was measured in six wells per group for each experiment on BeWo cells plated in 24-well plates. Each experiment was repeated at least twice with similar results, and representative results are shown.

Treatment of BeWo cells with phorbol ester and calcium ionophore. Stock solutions of phorbol ester and calcium ionophore were prepared in dimethyl sulfoxide (DMSO). These solutions were appropriately diluted with the culture medium and used for treatment of cells that had been pretreated with 1% fetal bovine serum for 18 h. The final concentration of DMSO during the treatment was <0.02%. For each experiment, control cells were treated with the respective concentration of DMSO. After incubation for the desired time, the medium was aspirated and cells were washed twice with the uptake buffer for the measurement of uptake or with phosphate-buffered saline for Northern blot and immunoblot analysis.

Northern blot analysis. Northern blot analysis was performed as described previously (8, 9, 35). After total RNA was isolated, polyadenylated RNA was prepared using the PolyATtract mRNA Isolation System (Promega, Madison, WI). Poly(A)+ RNA samples (2.0 µg/lane) were denatured, electrophoresed on 1.2% agarose-formaldehyde gels, and transferred to nylon membrane filters (Zeta-Probe, Bio-Rad, Richmond, CA) in 20× SSC (333 mmol/l NaCl and 333 mmol/l sodium citrate, pH 7.0), and the filters were baked at 80°C for 2 h. The filters were then prehybridized for 1 h at 43°C in hybridization buffer (50% formamide, 170 mmol/l Na2HPO4, 60 mmol/l NaH2PO4, 250 mmol/l NaCl, 1 mmol/l EDTA, and 7% SDS) and hybridized to the radiolabeled complementary 4F2hc or E16 (22) DNA (cDNA) probe (2 × 106 cpm/ml) for 18 h at 43°C. E16 is a truncated form of human LAT1 protein (33), and E16 cDNA can be used in Northern blot analysis for the detection of hLAT1 mRNA. Filters were washed for 10 min twice in 2× SSC-0.1% SDS at room temperature and then for 15 min in 1× SSC-0.1% SDS at 65°C and finally autoradiographed for 2 days at -80°C. The same blots were reprobed with beta -actin cDNA as a control to correct for the integrity and amount of RNA loaded. The band intensities were analyzed with a densitometer (Imaging Research, St. Catharine, ON, Canada), and the amounts of 4F2hc and hLAT1 mRNA were divided by the amounts of beta -actin mRNA in the same lanes.

Preparation of membrane fractions. Full-term placentas (37-41 wk of pregnancy) from uncomplicated pregnancies were obtained after cesarean section, and midtrimester placentas (12-21 wk) were obtained after artificial abortions with the informed consent of each patient. After removal of the cord, amniochorion, and decidual layer, the tissue was cut into small pieces and washed with 154 mmol/l NaCl to remove blood. The tissue was homogenized in 10 volumes of sucrose buffer containing 10 mmol/l Tris · HCl (pH 7.4), 250 mmol/l sucrose, 5 mmol/l EDTA, 1 mmol/l phenylmethylsulfonyl fluoride, 20 mg/l leupeptin, and 20 mg/l aprotinin with a Dounce homogenizer. The homogenized tissue was centrifuged at 2,600 rpm for 10 min at 4°C and the supernatant at 45,000 rpm for 1 h at 4°C. The pellets were resuspended in resuspension buffer containing 20 mmol/l Tris · HCl (pH 7.4), 1 mmol/l EDTA, 100 mmol/l NaCl, and 4 mmol/l MgCl2 (29) and stored at -20°C. The concentration of protein was determined using a Bradford protein assay kit (Bio-Rad) with BSA as the standard.

Purification of 4F2 complex protein, isolation of hybridoma cell line, and characterization of monoclonal antibody recognizing hLAT1. IMAA (GenBank AB040413, BAB20039) is a putative protein that consists of 180 amino acid residues and whose mRNA is expressed in HeLa S3 cells. Homology search analysis using advanced BLAST indicated that the amino acid sequence of IMAA has 92.2% homology with that of the NH2 terminus (180 residues) of hLAT1. It is assumed that IMAA corresponds to the three transmembrane domains of hLAT1 and that it may form an intermolecular disulfide bond with 4F2hc at Cys164. A cDNA expression vector that expresses the NH2-terminal region of IMAA (68 amino acid residues: NH2-MAGAGPKRRALAAPVAEEKEEAREKIMA- AKRADGAAPAGEGEGVTLQGNITLLKGVAVIVVAIMGSGI) was transformed into Escherichia coli strain BL21 (DE3) pLysS. After cultivation, the bacteria were harvested, and an inclusion body preparation was made. The bacterially expressed NH2-terminal amino acid fragment of IMAA in this crude inclusion body preparation was further purified by SDS-PAGE before injection into a BALB/c mouse. The purified IMAA fragment was mixed with 100 µg of lipopolysaccharide and injected subcutaneously into a BALB/c mouse. The mouse was boosted twice by such injections, and hybridoma cells were produced as described previously (45). Hybridoma cells of interest were further screened by immunoblot analysis (44) in which the plasma membrane fraction and purified 4F2 complex were used. Plasma membranes were prepared from HeLa S3 cells according to the method described by Maeda et al. (22). The plasma membranes were solubilized as described previously (30) and subjected to gel filtration chromatography to obtain the 4F2hc complex. The fractions were screened for 4F2hc antigen by dot-blot immunostaining using monoclonal antibody directed against 4F2hc (6-1-13) (43), and the antigen-positive fractions were applied to an immunoaffinity column (coupled with monoclonal antibody 6-1-13). The bound 4F2 complex purified from HeLa S3 cells, which should contain the heterodimer of 4F2hc and hLAT1, was separated by SDS-PAGE using a Tris-glycine buffer system under reducing conditions (39) and blotted onto a nitrocellulose membrane. The membrane was treated successively with monoclonal antibody (181C) or the monoclonal antibody against human parainfluenza virus type 2F protein as a negative control (117-1A) (44), biotinylated horse immunoglobulin to mouse IgG (heavy and light chains; Vector Laboratories, Burlingame, CA), and avidin-biotin-peroxidase complex (Vector Laboratories). The immunostained bands were visualized as described previously (44). This monoclonal antibody (181C) detected a 40-kDa protein in a 4F2 complex consisting of 4F2hc and hLAT1, and similar results of immunoblot analysis were obtained for the membrane fraction of HeLa S3 cells (data not shown). For competition studies, we used a synthetic peptide consisting of the 36-46th amino acids from the NH2 terminus of IMAA (APAGEGEGVTL) as the blocking peptide for 181C.

Immunoblot analysis. The proteins were denatured with an equal amount of buffer containing 200 mmol/l dithiothreitol, 20% glycerol, 0.04% bromphenol blue, 10% SDS, and 120 mmol/l Tris · HCl (pH 6.8) and incubated for 5 min at 100°C. Denatured samples (50 µg/lane) were subjected to 7.5% SDS-PAGE (21) and transferred to nitrocellulose filters (46) (Bio-Rad).

The membranes were presoaked in 5% nonfat dry milk in 10 mmol/l Tris-buffered saline (TBS) for 1.5 h at room temperature. They were then incubated with anti-4F2hc goat polyclonal antibody (1:1,600; Research Diagnostics, Flanders, NJ) in 5% nonfat dry milk in TBS-T (0.1% Tween 20 in TBS), with the anti-4F2hc antibody that had been preincubated with the blocking peptide (Research Diagnostics), with the undiluted culture fluid containing the monoclonal antibody (181C), or with the monoclonal antibody (181C) that had been preincubated with the synthetic blocking peptide (APAGEGEGVTL) for 181C for 2 h at room temperature. The membranes were washed three times with TBS-T and then incubated with peroxidase-labeled rabbit anti-goat IgG (1: 10,000; Kirkegaard and Perry Laboratories, Gaithersburg, MD) or with peroxidase-labeled goat anti-mouse IgG (1: 15,000; Promega) for 30 min at room temperature and developed for the detection of the specific protein using enhanced chemiluminescence reagents (Amersham). The band intensities were analyzed as described above.

Immunohistochemistry. Representative blocks of formalin-fixed, paraffin-embedded placental tissue were cut to 4-µm-thick sections and processed by the standard avidin-biotin peroxidase method. Briefly, the sections were dewaxed in xylene and rinsed in ethanol and a graded series of ethanol in water, and the endogenous peroxidase activity was blocked with 3% hydrogen peroxide. Samples were incubated with 1% bovine serum and then with the primary antibody [the polyclonal antibody to 4F2hc, the monoclonal antibody to cytokeratin AE1/AE3 (DAKO, Carpinteria, CA) at 1 mg/l, or the culture fluid containing the monoclonal antibody (181C) for detection of hLAT1] for 2 h at room temperature. After the samples were rinsed, biotinylated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and horseradish peroxidase-conjugated streptavidin (Nichirei, Tokyo, Japan) were applied to the sections according to the manufacturer's instructions. Peroxidase activity was visualized by means of a 3-min application of diaminobenzidine chromogen containing 0.05% hydrogen peroxide. The sections were then counterstained with hematoxylin, dehydrated, cleared, and mounted. Control sections were also treated with the anti-4F2hc antibody preincubated with a blocking peptide or with 181C preincubated with a blocking peptide.

Statistical methods. Values are means ± SE and were statistically analyzed by analysis of variance followed by the unpaired t-test. Statistical significance was accepted at P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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It has been shown that hLAT1, when combined as a heterodimer with 4F2hc, is capable of amino acid transport activity characteristic of system L. Northern blot analysis demonstrated that the mRNAs encoding these proteins were expressed in the human placenta and a BeWo human choriocarcinoma cell line (Fig. 1).


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Fig. 1.   Northern blot analysis of 4F2hc and hLAT1 mRNAs in human placenta and a human choriocarcinoma BeWo cell line. Poly(A)+ RNA (2 µg/lane) from human full-term placenta (lane 1) and BeWo cells (lane 2) was electrophoresed and hybridized with 32P-labeled 4F2hc (top) or hLAT1 (bottom) cDNA probe. Positions of 28S and 18S RNA are indicated at right. Data are from a representative experiment that was repeated 3 times with similar results.

To examine the presence of 4F2hc and hLAT1 proteins, we performed immunoblot analysis of the 4F2hc and hLAT1 proteins in human placenta and the BeWo cell line. Figure 2A shows that these proteins were detected in the BeWo cell line (lane 1) and human placenta (lane 3). When the blots were incubated with the antibody to 4F2hc that had been treated with a blocking peptide (lanes 2 and 4, top) or with 181C that had been treated with a blocking peptide (lanes 2 and 4, bottom) as negative controls, these protein signals were not detected. Next, we used immunoblot analysis of the 4F2hc and hLAT1 proteins in the human placenta to detect gestational changes in these proteins. Densitometric scanning of the immunoblots showed that the level of 4F2hc protein in the full-term placenta (2.32 ± 0.51 arbitrary units) was significantly (P < 0.05) higher than that in the midtrimester placenta (0.94 ± 0.40 arbitrary units). Similar results were observed for the levels of hLAT1 protein in the midtrimester (0.48 ± 0.26 arbitrary units) and the full-term (0.84 ± 0.13 arbitrary units) placenta (Fig. 2B).


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Fig. 2.   Immunoblot analysis of 4F2hc and hLAT1 proteins in human placenta and human choriocarcinoma BeWo cell line. A: protein (50 µg/lane) extracted from BeWo cells (lanes 1 and 2) or human full-term placenta (lanes 3 and 4) was electrophoresed on a 7.5% SDS-polyacrylamide gel and transferred to a nitrocellulose filter. Filter was incubated with goat anti-4F2hc antibody (lanes 1 and 3, top), with the monoclonal antibody 181C (lanes 1 and 3, bottom), with the anti-4F2hc antibody preincubated with a blocking peptide (lanes 2 and 4, top), or with 181C treated with a blocking peptide (lanes 2 and 4, bottom). Positions of molecular markers are indicated at right. B: filter containing protein (50 µg/lane) extracted from midtrimester placentas (lanes 1-3) or full-term placentas (lanes 4-6) was treated with goat anti-4F2hc antibody (top) or 181C (bottom) as described for A. Data are from a representative experiment that was repeated 3 times with similar results.

Because it is known that the localization of the amino acid transport systems is polarized in the human placenta, we used immunohistochemistry to detect 4F2hc and hLAT1 and found that these proteins were localized mainly in the placental apical membrane (Fig. 3, A and B). We also detected endothelial staining (Fig. 3, C and E), which seemed weaker than that in the apical membrane. When the sections were treated with anti-4F2hc antibody after preincubation with the blocking peptide, the immunostaining was not detected (Fig. 3D). When control sections were treated with 181C preincubated with the blocking peptide, no noticeable staining was observed (Fig. 3F). When the sections were treated with antibody against cytokeratin, staining was observed not in the membrane but in the cytosol (data not shown).


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Fig. 3.   Immunohistochemical localization of 4F2hc and hLAT1 in human full-term placenta. A and B: immunohistochemical localization of 4F2hc and hLAT1, respectively, in paraffin sections (4 µm thick) of normal human full-term placenta at 38 wk of pregnancy; 4F2hc and hLAT1 were localized mainly in the placental apical membrane. C and E: endothelial staining of 4F2hc and hLAT1, respectively. Control sections were treated with the anti-4F2hc antibody preincubated with a blocking peptide (D) or with 181C preincubated with a blocking peptide (F). Final magnification, ×400. Scale bar, 10 µm.

It has been reported that treatment with PMA stimulates the system L transport activity in a human choriocarcinoma cell line (JAR) (34) and that combined treatment with PMA and calcium ionophore stimulates the expression of hLAT1 mRNA in lymphoid cells (12). We therefore conducted [3H]leucine uptake experiments on BeWo cells after PMA and calcium ionophore treatment to determine whether the activity of system L was regulated by these effectors in these cells. The uptake of leucine into these cells was linear for >= 10 min (data not shown), and therefore, we measured the [3H]leucine uptake into the cells for 5 min. Although treatment with PMA (1 µmol/l) or calcium ionophore (1 µmol/l) alone did not stimulate leucine uptake, combined treatment with PMA and calcium ionophore for 3 h significantly (P < 0.01) stimulated it (Fig. 4A). Stimulation was also observed after 6 h, but not after 1 h, of treatment. Although the uptake of leucine was decreased 18% (Fig. 4B) when the uptake buffer was changed to choline chloride instead of sodium chloride, stimulation of leucine uptake by PMA and calcium ionophore was similar in both buffers. To determine the substrate specificity of the transport system responsible for leucine uptake in these cells, we studied the effects of unlabeled BCH, a model substrate for the L system, on the uptake of leucine. BCH (1 mmol/l) caused 90% inhibition of leucine uptake (Fig. 4B), and no stimulation of leucine uptake by PMA and calcium ionophore was observed in the presence of BCH.


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Fig. 4.   Time-dependent effects of phorbol 12-myristate 13-acetate (PMA) and calcium ionophore (A-23187) on leucine uptake in BeWo cells. A: BeWo cells (1.0 × 105 cells/well) were incubated with 1 µmol/l PMA and/or 1 µmol/l calcium ionophore. Amount of [3H]leucine taken up by cells during 5 min was measured in each well. B: amount of [3H]leucine taken up by cells over 5 min was measured in medium containing sodium chloride (Na+), choline chloride (Na-), or unlabeled 2-amino-2-norbornane-carboxylic acid (BCH+). Concentration of [3H]leucine was 10 nmol/l, and that of BCH was 1 mmol/l. Values are means ± SE from 6 wells per group for each experiment. To determine statistical significance, values for the indicated period were compared with those for the corresponding control. *P < 0.01 vs. control (unpaired t-test).

To determine whether the increase in leucine uptake in the BeWo cells was due to increases in 4F2hc and/or hLAT1 expression, we first used Northern blot analysis to analyze the changes in the steady-state levels of 4F2hc and hLAT1 mRNAs. Combined treatment with PMA and calcium ionophore significantly (P < 0.05) increased the levels of 4F2hc and hLAT1 mRNAs, while the amount of beta -actin mRNA did not change significantly after the treatment (normalized band intensities in arbitrary units: 0.89 ± 0.33 for 4F2hc control, 6.07 ± 1.56 after PMA and calcium ionophore treatment, 0.55 ± 0.22 for hLAT1 control, and 1.69 ± 0.51 after PMA and calcium ionophore treatment; Fig. 5A), which was consistent with the results for leucine uptake shown in Fig. 4. Next, we used immunoblot analysis of the 4F2hc and hLAT1 proteins in the BeWo cells to analyze the changes of these proteins. Densitometric scanning showed that combined treatment with PMA and calcium ionophore significantly (P < 0.01) increased the levels of 4F2hc and hLAT1 proteins (Fig. 5B).


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Fig. 5.   Effects of PMA and calcium ionophore on expression of 4F2hc and hLAT1 mRNA and protein in BeWo cells. A: Northern blot analysis of 4F2hc and hLAT1 mRNAs in control BeWo cells (lanes 1 and 2) and BeWo cells treated with PMA and calcium ionophore (lanes 3 and 4). Poly(A)+ RNA (2 µg/lane) was prepared from BeWo cells treated or not treated with PMA and calcium ionophore for 3 h and analyzed by hybridization with 32P-labeled 4F2hc, hLAT1, or beta -actin cDNA probe. Data are from a representative experiment that was repeated 3 times with similar results. B: immunoblot analysis of 4F2hc and hLAT1 proteins in control BeWo cells and BeWo cells treated with PMA and calcium ionophore. Filters containing protein (50 µg/lane) extracted from cells treated (open bars) or not treated (solid bars) with PMA and calcium ionophore for 3 h were treated with goat anti-4F2hc antibody or the monoclonal antibody 181C (see MATERIALS AND METHODS). Band intensities were analyzed with a densitometer, and data are expressed as normalized band intensity in arbitrary units. Values are means ± SE. *P < 0.01 vs. control (unpaired t-test).


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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Heterodimers of 4F2hc and light chains such as LAT1, y+LAT1, y+LAT2, xCT, and LAT2 have recently been shown to be responsible for several systems of amino acid transport. The heterodimer of 4F2hc and LAT1 in particular is thought to function as a system L amino acid transporter that can transfer neutral and aromatic amino acids in a sodium-independent manner. In previous reports, the expression of 4F2hc, LAT1 (18), and LAT2 (32, 40) mRNAs in human placenta was shown. The present study demonstrated the expression of 4F2hc and hLAT1 mRNA in the human placenta and a human choriocarcinoma (BeWo) cell line by means of Northern blot analysis with findings similar to those reported previously (20, 33).

The 4F2hc and hLAT1 proteins were also detected in the human placental membrane by immunoblot analysis. Together, these results suggest that the 4F2hc-hLAT1 heterodimer works as a system L amino acid transporter in the placenta in a manner similar to that in other organs. Although apical and basal membranes were included in our experiment, the levels of 4F2hc and hLAT1 proteins increased at full term compared with those at midtrimester (Fig. 2B), which agrees with the results of a previous study of rat placenta (27). It seems likely that the increased levels of placental 4F2hc and hLAT1 proteins in the L system play an important role in amino acid transport from mother to fetus during pregnancy.

In the immunoblot analysis of hLAT1 protein, we detected a protein band of ~50 kDa in placenta and one of ~40 kDa in BeWo cells. Although we do not now why the apparent molecular size of the placental hLAT1 protein in our immunoblot analysis was larger than expected, the discrepancy may be the result of differences in posttranscriptional regulation of hLAT1 mRNA or posttranscriptional modification of hLAT1 protein, perhaps related to the glycoprotein characteristics of hLAT1 protein (33). In studies using an anti-GLUT-1 antibody, we made a similar observation that the molecular size of GLUT-1 protein detected by immunoblot analysis differs between human placental tissue [60 kDa (minor band) and 49 kDa (major band)] (36) and BeWo cells (broad 46 kDa) (28).

Next we examined whether any significant changes occur in the levels of 4F2hc or hLAT1 mRNA in human placenta between midtrimester and full term, but none were detected (data not shown). This discrepancy between the results of immunoblot and Northern blot analysis may be due to possible posttranscriptional regulation of the 4F2hc and hLAT1 mRNAs or modification of the 4F2hc and hLAT1 proteins.

In the human placenta, the amino acid transport systems are thought to be predominantly located in specific membranes (15-17, 34). Therefore, apical and basal membranes from the human placenta were analyzed separately to examine which membrane contains which specific amino acid transport system(s) on the basis of findings that apical and basal membranes possess system L (15-17) and y+L (3, 11) amino acid transport. It was not known whether the apical or basal membrane expresses heterodimers composed of the 4F2hc protein in combination with hLAT1, hLAT2, or y+LAT to form the transporter for the L or y+L system, respectively. Our immunohistochemical data revealed that specific staining for 4F2hc and hLAT1, which are responsible for system L amino acid transport, was localized in the apical, but not the basal, membrane of trophoblasts. It was reported previously (3) that 4F2hc proteins were not detected in the basal membrane, despite functional evidence for the presence of system y+L. Our data about 4F2hc protein localization are consistent with those reported by Ayuk et al. (3).

To our knowledge, only a few studies of the regulation of amino acid transport system L have been published. In one previous study, it was shown that treatment of JAR human choriocarcinoma cells with PMA resulted in a significant stimulation of system L as measured in terms of radiolabeled amino acid uptake into the cells (34). Another study demonstrated that E16 cDNA, a truncated form of hLAT1 cDNA, could be cloned by the subtraction hybridization method after stimulation of the lymphoid cells with PMA and calcium ionophore (12). However, there are no reports on how the system L amino acid transporter is regulated with respect to 4F2hc and hLAT1 in the human placenta. We therefore examined the effects of PMA and calcium ionophore treatment on the uptake of leucine (a representative amino acid transported by system L) and the expression of 4F2hc and hLAT1 mRNAs and proteins in a human choriocarcinoma (BeWo) cell line. We chose BeWo cells, which have the potential for differentiation, because it has been shown that the cells undergo fusion and morphological differentiation after cAMP treatment similar to the fusion of cytotrophoblasts to form syncytiotrophoblasts after cAMP treatment. Combined treatment with PMA and calcium ionophore resulted in significantly increased uptake of leucine in the BeWo cells. We also demonstrated that incubation of BeWo cells with PMA and calcium ionophore induced a significant increase in 4F2hc and hLAT1 mRNAs and proteins. These data are not consistent with previously reported data which indicated that the expression of 4F2hc, not of hLAT1, may be rate limiting for system L amino acid transport (20). This discrepancy may result from differences between the cell line and tissue. The published study used dissected pieces of chorionic villi from full-term placenta. Additionally, E16 mRNA (a truncated form of hLAT1 mRNA) was increased in lymphoid cells after the stimulation with PMA and calcium ionophore (11), which was similar to our findings. Figure 4B shows that BCH (1 mmol/l), a model substrate for the L system, did not completely inhibit the uptake of leucine. This suggests that uptake of leucine into the cells is also mediated by other transport systems. Moreover, we showed previously that the degree of the increase of GLUT-1 mRNA was greater than that of 2-deoxyglucose uptake in BeWo cells after cAMP treatment (28). The discrepancy between the changes in the amino acid uptake level and the mRNA level for system L may arise from similar causes. These results suggest that PMA and calcium ionophore may play an important role in the regulation of leucine uptake mediated by system L and in 4F2hc and hLAT1 mRNA and protein expression in BeWo cells. The molecular mechanisms that are responsible for the specific stimulation of leucine uptake and 4F2hc and hLAT1 expression by PMA and calcium ionophore in BeWo cells are not fully understood. PMA is a phorbol ester that is capable of activating protein kinase C, and PMA has been shown to have a stimulating effect on human chorionic gonadotropin secretion in choriocarcinoma cells (1). It has also been suggested that calcium ionophores, which increase intracellular calcium, enhance the potency of phorbol ester for activating protein kinase C in human leukemia cells (25) and in Madin-Darby canine kidney cells (47). We showed that PMA and intracellular calcium synergistically stimulate the expression of 4F2hc and hLAT1 mRNAs and proteins. These events may be involved in regulating the levels of leucine uptake after PMA and calcium ionophore treatment.

Our results suggest that the levels of 4F2hc and hLAT1 may regulate the system L amino acid transport in the human placenta. Further investigations are needed to clarify the molecular mechanisms responsible for the expression and regulation of the 4F2hc-LAT1 heterodimer and other amino acid transporters in the human placenta.


    FOOTNOTES

Address for reprint requests and other correspondence: M. Sakata, Dept. of Obstetrics and Gynecology, Osaka University Faculty of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan (E-mail: msakata{at}gyne.med.osaka-u.ac.jp).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 18 December 2000; accepted in final form 4 September 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Andersen, B, Milsted A, Kennedy G, and Nilson JH. Cyclic AMP and phorbol esters interact synergistically to regulate expression of the chorionic gonadotropin genes. J Biol Chem 263: 15578-15583, 1988[Abstract/Free Full Text].

2.   Asano, T, Shibasaki Y, Kasuga M, Kanazawa Y, Takaku F, Akanuma Y, and Oka Y. Cloning of a rabbit brain glucose transporter cDNA and alteration of glucose transporter mRNA during tissue development. Biochem Biophys Res Commun 154: 1204-1211, 1988[ISI][Medline].

3.   Ayuk, PT, Sibley CP, Donnai P, D'Souza S, and Glazier JD. Development and polarization of cationic amino acid transporters and regulators in the human placenta. Am J Physiol Cell Physiol 278: C1162-C1170, 2000[Abstract/Free Full Text].

4.   Baly, DL, and Horuk R. The biology and biochemistry of the glucose transporter. Biochim Biophys Acta 947: 571-590, 1988[ISI][Medline].

5.   Bell, GI, Kayano T, Buse JB, Burant CF, Takeda J, Lin D, Fukumoto H, and Seino S. Molecular biology of mammalian glucose transporters. Diabetes Care 13: 198-208, 1990[Abstract].

6.   Bloxam, DL, Bax BE, and Bax CMR Epidermal growth factor and insulin-like growth factor I differently influence the directional accumulation and transfer of 2-aminoisobutyrate (AIB) by human placental trophoblast in two-sided culture. Biochem Biophys Res Commun 199: 922-929, 1994[ISI][Medline].

7.   Brandsch, M, Leibach FH, Mahesh VB, and Ganapathy V. Calmodulin-dependent modulation of pH sensitivity of the amino acid transport system L in human placental choriocarcinoma cells. Biochim Biophys Acta 1192: 177-184, 1994[ISI][Medline].

8.   Chen, CF, Kurachi H, Fujita Y, Terakawa N, Miyake A, and Tanizawa O. Changes in epidermal growth factor receptor and its messenger ribonucleic acid levels in human placenta and isolated trophoblast cells during pregnancy. J Clin Endocrinol Metab 67: 1171-1177, 1988[Abstract].

9.   Chomczynski, P, and Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156-159, 1987[ISI][Medline].

10.   Christensen, HN, Albritton LM, Kakuda DK, and MacLeod CL. Gene-product designations for amino acid transporters. J Exp Biol 196: 51-57, 1994[Abstract/Free Full Text].

11.   Furesz, TC, and Smith CH. Identification of two leucine-sensitive lysine transport activities in human placental basal membrane. Placenta 18: 649-655, 1997[ISI][Medline].

12.   Gaugitsch, HW, Prieschl EE, Kalthoff F, Huber NE, and Baumruker T. A novel transiently expressed, integral membrane protein linked to cell activation. Molecular cloning via the rapid degradation signal AUUUA. J Biol Chem 267: 11267-11273, 1992[Abstract/Free Full Text].

13.   Haynes, BF, Hemler ME, Mann DL, Eisenbarth GS, Shelhamer J, Mostowski HS, Thomas CA, Strominger JL, and Fauci AS. Characterization of a monoclonal antibody. J Biol Chem 273: 23629-23632, 1998[Abstract/Free Full Text].

14.   Hemler, ME, and Strominger JL. Characterization of antigen recognized by the monoclonal antibody (4F2): different molecular forms on human T and B lymphoblastoid cell lines. J Immunol 129: 623-628, 1982[Abstract/Free Full Text].

15.   Hoeltzli, SD, and Smith CH. Alanine transport systems in isolated basal plasma membrane of human placenta. Am J Physiol Cell Physiol 256: C630-C637, 1989[Abstract/Free Full Text].

16.   Illsley, NP, Wang Z-Q, Gray A, Sellers MC, and Jacobs MM. Simultaneous preparation of paired, syncytial, microvillous and basal membranes from human placenta. Biochim Biophys Acta 1029: 218-226, 1990[ISI][Medline].

17.   Johnson, LW, and Smith CH. Neutral amino acid transport systems of microvillous membrane of human placenta. Am J Physiol Cell Physiol 254: C773-C780, 1988[Abstract/Free Full Text].

18.   Kanai, Y, Segawa H, Miyamoto K, Uchino H, Takeda E, and Endou H. Expression cloning and characterization of a transporter for large neutral amino acids activated by the heavy chain of 4F2 antigen (CD98). J Biol Chem 273: 23629-23632, 1998[Abstract/Free Full Text].

19.   Karl, PI, Alpy KL, and Fisher SE. Amino acid transport by the cultured human placental trophoblast: effect of insulin on AIB transport. Am J Physiol Cell Physiol 262: C834-C839, 1992[Abstract/Free Full Text].

20.   Kudo, Y, and Boyd CAR Heterodimeric amino acid transporters: expression of heavy but not light chains of CD98 correlates with induction of amino acid transport systems in human placental trophoblast. J Physiol (Lond) 523: 13-18, 2000[Abstract/Free Full Text].

21.   Laemmli, UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 277: 680-685, 1970.

22.   Maeda, T, Balakrishnan K, and Mehdi O. A simple and rapid method for the preparation of plasma membranes. Biochim Biophys Acta 731: 115-120, 1983[ISI][Medline].

23.   Mannion, BA, Kolesnikova TV, Lin SH, Wang S, Thompson NL, and Hemler ME. The light chain of CD98 is identified as E16/TA1 protein. J Biol Chem 273: 33127-33129, 1998[Abstract/Free Full Text].

24.   Mastroberardino, L, Spindler B, Pfeiffer R, Skelly PJ, Loffing J, Shoemaker CB, and Verrey F. Amino-acid transport by heterodimers of 4F2hc/CD98 and members of a permease family. Nature 395: 288-291, 1998[ISI][Medline].

25.   May, WS, Jr, Sahyoun N, Wolf M, and Cuatrecasas P. Role of intracellular calcium mobilization in the regulation of protein kinase C-mediated membrane processes. Nature 317: 549-551, 1985[ISI][Medline].

26.   Nakamura, E, Sato M, Yang H, Miyagawa F, Harasaki M, Tomita K, Matsuoka S, Noma A, Iwai K, and Minato N. 4F2 (CD98) heavy chain is associated covalently with an amino acid transporter and controls intracellular trafficking and membrane topology of 4F2 heterodimer. J Biol Chem 274: 3009-3016, 1999[Abstract/Free Full Text].

27.   Novak, DA, Matthews JC, Beveridge MJ, Yao SY, Young J, and Kilberg MS. Demonstration of system y+L activity on the basal plasma membrane surface of rat placenta and developmentally regulated expression of 4F2HC mRNA. Placenta 18: 643-648, 1997[ISI][Medline].

28.   Ogura, K, Sakata M, Okamoto Y, Yasui Y, Tadokoro C, Yoshimoto Y, Yamaguchi M, Kurachi H, Maeda T, and Murata Y. 8-Bromo-cyclic AMP stimulates glucose transporter-1 expression in a human choriocarcinoma cell line. J Endocrinol 164: 171-178, 2000[Abstract/Free Full Text].

29.   Ogura, K, Sakata M, Yamaguchi M, Kurachi H, and Murata Y. High concentration of glucose decreases glucose transporter-1 expression in mouse placenta in vitro and in vivo. J Endocrinol 160: 443-452, 1999[Abstract/Free Full Text].

30.   Ohta, H, Tsurudome M, Matsumura H, Koga Y, Morikawa S, Kawano M, Kusagawa S, Komada H, Nishio M, and Ito Y. Molecular and biological characterization of fusion regulatory proteins (FRPs): anti-FRP mAbs induced HIV-mediated cell fusion via an integrin system. EMBO J 13: 2044-2055, 1994[Abstract].

31.   Pfeiffer, R, Rossier G, Spindler B, Meier C, Kuhn L, and Verrey F. Amino acid transport of y+L-type by heterodimers of 4F2hc/CD98 and members of the glycoprotein-associated amino acid transporter family. EMBO J 18: 49-57, 1999[Abstract/Free Full Text].

32.   Pineda, M, Fernandez E, Torrents D, Estevez R, Lopez C, Camps M, Lloberas J, Zorzano A, and Palacin M. Identification of a membrane protein, LAT-2, that co-expresses with 4F2 heavy chain, an L-type amino acid transport activity with broad specificity for small and large zwitterionic amino acids. J Biol Chem 274: 19738-19744, 1999[Abstract/Free Full Text].

33.   Prasad, PD, Wang H, Huang W, Kekuda R, Rajan DP, Leibach FH, and Ganapathy V. Human LAT1, a subunit of system L amino acid transporter: molecular cloning and transport function. Biochem Biophys Res Commun 255: 283-288, 1999[ISI][Medline].

34.   Ramamoorthy, S, Leibach FH, Mahesh VB, and Ganapathy V. Modulation of the activity of amino acid transport system L by phorbol esters and calmodulin antagonists in a human placental choriocarcinoma cell line. Biochim Biophys Acta 1136: 181-188, 1992[ISI][Medline].

35.   Sakata, M, Farooqui SM, and Prasad C. Post-transcriptional regulation of loss of rat striatal D2 dopamine receptor during aging. Brain Res 575: 309-314, 1992[ISI][Medline].

36.   Sakata, M, Kurachi H, Imai T, Tadokoro C, Yamaguchi M, Yoshimoto Y, Oka Y, and Miyake A. Increase in human placental glucose transporter-1 during pregnancy. Eur J Endocrinol 132: 206-212, 1995[ISI][Medline].

37.   Sakata, M, Yamaguchi M, Imai T, Tadokoro C, Yoshimoto Y, Oka Y, Kurachi H, and Miyake A. 8-Bromo-cAMP inhibits glucose transport activity in mouse placental cells in culture. J Endocrinol 150: 319-327, 1996[Abstract].

38.   Sato, H, Tamba M, Ishii T, and Bannai S. Cloning and expression of a plasma membrane cystine/glutamate exchange transporter composed of two distinct proteins. J Biol Chem 274: 11455-11458, 1999[Abstract/Free Full Text].

39.   Schägger, H, and von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166: 368-379, 1987[ISI][Medline].

40.   Segawa, H, Fukasawa Y, Miyamoto K, Takeda E, Endou H, and Kanai Y. Identification and functional characterization of an Na+-independent neutral amino acid transporter with broad substrate selectivity. J Biol Chem 274: 19745-19751, 1999[Abstract/Free Full Text].

41.   Tadokoro, C, Yoshimoto Y, Sakata M, Fujimiya M, Kurachi H, Adachi E, Maeda T, and Miyake A. Localization of human placental glucose transporter 1 during pregnancy: an immunohistochemical study. Histol Histopathol 11: 673-681, 1996[ISI][Medline].

42.   Torrents, D, Estevez R, Pineda M, Fernandez E, Lloberas J, Shi YB, Zorzano A, and Palacin M. Identification and characterization of a membrane protein (y+L amino acid transporter-1) that associates with 4F2hc to encode the amino acid transport activity y+L: a candidate gene for lysinuric protein intolerance. J Biol Chem 273: 32437-32445, 1998[Abstract/Free Full Text].

43.   Tsurudome, M, Ito M, Takebayashi S, Okumura K, Nishio M, Kawano M, Kusagawa S, Komada H, and Ito Y. Cutting edge: primary structure of the light chain of fusion regulatory protein-1/ CD98/4F2 predicts a protein with multiple transmembrane domains that is almost identical to the amino acid transporter E16. J Immunol 162: 2462-2466, 1999[Abstract/Free Full Text].

44.   Tsurudome, M, Nishio M, Komada H, Bando H, and Ito Y. Extensive antigenic diversity among human parainfluenza virus type 2 virus isolates and immunological relationship among paramyxoviruses revealed by monoclonal antibodies. Virology 171: 38-48, 1989[ISI][Medline].

45.   Tsurudome, M, Yamada A, Hishiyama M, and Ito Y. Monoclonal antibodies against the glycoproteins of mumps virus: fusion inhibition by anti-HN monoclonal antibody. J Gen Virol 67: 2259-2265, 1986[Abstract].

46.   Wilson, PT, Gershoni JM, Hawrot E, and Lentz TL. Binding of alpha -bungarotoxin to proteolytic fragments of the alpha -subunit of Torpedo acetylcholine receptor analyzed by protein transfer on positively charged membrane filters. Proc Natl Acad Sci USA 81: 2553-2557, 1984[Abstract].

47.   Yokota, K. Cellular mechanism of synergistic stimulation of PGE2 production by phorbol diester and Ca2+ ionophore A23187 in cultured Madin-Darby canine kidney cells. Arch Biochem Biophys 288: 192-201, 1991[ISI][Medline].


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