In vitro study of placental trophoblast calcium uptake using JEG-3 human

choriocarcinoma cells

ROCKY S. TUAN*, CLAIRE J. MOORE, JACQUELINE W. BRITTINGHAM, JAMES J. KIRWIN,

ROBERT E. AKINS and MAYME WONG

Department of Orthopaedic Surgery, Thomas Jefferson University, 1015 Walnut Street, Philadelphia, PA 19107, USA

* Author for correspondence

Summary

During human fetal development, placental syncytio-trophoblasts actively transport calcium from thematernal to the fetal circulation. Two functionalcomponents, a cytosolic Ca2+-binding protein (CaBP)and a Ca2+-ATPase have been identified in thesyncytdotrophoblasts of the chorionic villi. We reporthere the calcium uptake properties of a humanchoriocarcinoma cell line, JEG-3, which was used asan in vitro model cell system for the syncytiotro-phoblasts. In culture, JEG-3 proliferated as largesyncytial aggregates expressing typical syncytio-trophoblast markers. 48Ca uptake by JEG-3 was asubstrate- and temperature-dependent, membrane-mediated active process that exhibited linear kin-etics for up to 7min. Both the CaBP and the Ca2+-ATPase were expressed by JEG-3, on the basis ofbiochemical, histochemical, immunochemical and/ormRNA asssays. Immunohistochemistry and in situhybridization revealed that JEG-3 cells were hetero-geneous with respect to the expression of the CaBP.The Ca2+-ATPase activity of JEG-3 was similar to theplacental enzyme in terms of sensitivity to specificinhibitors, and was detected histochemically along

the cell membrane. Fura-2 Ca2+ imaging revealedthat calcium uptake by JEG-3 was not accompaniedby a concomitant increase in cytosolic [Ca2+], sugges-ting a specific Ca2+ sequestration mechanism. Theinvolvement of calciotropic hormonal regulation wasevaluated by studying the response of JEG-3 to 1,25-dihydroxy vitamin D3. Calcium uptake was signifi-cantly stimulated in a dose-dependent manner by a24-h treatment of the cells with 1,25-dihydroxyvitamin D3 (optimal dose —0.5 nM); the CaBP leveldoubled whereas steady-state CaBP mRNA did not,suggesting that CaBP expression was regulated by1,25-dihydroxy vitamin Da. These observationsstrongly suggest that the JEG-3 human choriocarci-noma cells should serve as a convenient in vitromodel system for studying the cellular mechanismand regulation of transplacental calcium transport.

Key words: calcium-binding protein, Ca2+-ATPase, membranetransport, placental calcium transport, immunohistochemistry,vitamin D, gene expression, embryonic development, in situhybridization.

Introduction

Mammalian fetal nutrition during development is whollydependent on the transport of nutrients by the placenta(Boyd, 1987; Hill and Longo, 1980; Munro et al. 1983;Sherman and Boyd, 1987; Truman and Ford, 1984).Calcium, needed for skeletal formation, neuromuscularfunctions, and other physiological activities, is trans-ported actively across the placenta from the maternal tothe fetal circulation (Brunette, 1988; Pitkin, 1985; vanKreel and van Dijk, 1983). This process is carried out bythe placental trophoblastic cells (Dearden and Ockleford,1983; Loke and Whyte, 1983), which line the chorionic villiand represent the epithelial layer separating the maternaland fetal circulations. In the human placental chorionicvilli, a layer of syncytiotrophoblasts lies over the cyto tro-phoblasts, and surrounds the internal mesoderm and fetalcapillaries (Ramsey, 1975). Calcium transport by pla-cental trophoblasts is therefore analogous to epithelialtransport in general, i.e. calcium is moved in a transcellu-lar manner. Furthermore, the calcium level is higher in

Journal of Cell Science 98, 333-342 (1991)Printed in Great Britain © The Company of Biologiats Limited 1991

the fetal circulation than in the maternal circulation. It iswell established that calcium is actively transportedagainst a concentration gradient by placental tropho-blasts.

Our laboratory has been studying the cellular andmolecular mechanism and regulation of placental calciumtransport (Tuan, 1982,1985; Tuan and Bigioni, 1990; Tuanand Cavanaugh, 1986; Tuan and Kirwin, 1988; Tuan andKushner, 1987; Tuan et al. 1988). These studies, which aresummarized below, have shown that mammalian pla-cental trophoblasts express two marker molecules that arefunctional components of the calcium transport mechan-ism: a specific, high-Afr calcium-binding protein (CaBP)and an integral membrane Ca2+-activated ATPase. Boththe CaBP and the Ca2+-ATPase are expressed as afunction of embryonic development in a manner thatparallels the onset of placental calcium transport. Thetrophoblastic localization of the CaBP and Ca2+-ATPasewas revealed by immunohistochemistry and enzymehistochemistry, respectively. The functional involvementof the CaBP and Ca -ATPase in transmembrane calcium

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transport was demonstrated using cell-free placentalmembrane vesicles, whose active, ATP-dependent calciumuptake was inhibited by antibodies directed against theCaBP and inhibitors of the Ca2+-ATPase. Recently, acDNA to the placental CaBP has been cloned, and hasbeen used to probe its gene expression with respect totemporal profile (Northern blot hybridization) and cellularlocation (in situ hybridization). Thus, CaBP mRNA isfound to increase in level during development, and isexpressed only by placental trophoblasts. These studieshave been carried out with both human and mouseplacentae, with essentially similar results.

Although much information on the physiological, mol-ecular and biochemical aspects of trophoblast calciumtransport has been obtained using whole placental tissue,the cellular mechanism of the transport process remainsunknown. The experimental means of analyzing transportare mostly limited to either whole organ perfusion (Sweiryet al. 1980; van Kreel and van Dijk, 1983) or uptake by cell-free vesicles (Bissonnette, 1982; Fisher et al. 1987; Shamiet al. 1975; Tuan, 1985; Whitsett and Tsang, 1980),whereas whole cell studies dealing with isolated primarytrophoblasts are made complicated by their varyinghomogeneity and availability (Contractor et al. 1984; Huntet al. 1989). As in other epithelial transport systems(Rodriguez-Boulan and Nelson, 1989), these problems maybe alleviated by identifying and studying a stable cell lineire vitro, perhaps analogous to the popular Madin-Darbycanine kidney (MDCK) cells. Towards this goal, we reporthere our findings on the characterization of a candidate irevitro cellular model of trophoblast calcium uptake, thehuman JEG-3 choriocarcinoma cells. Derived originallyfrom the BeWo human choriocarcinoma cell line, JEG-3cells in culture form large, multinucleated syncytia(Babalola et al. 1990), and express abundant humanchorionic gonadotropin and chorionic somatomammotro-pin, hallmarks of trophoblastic cells (Kohler and Bridson,1971; Patillo and Gey, 1968). The objective of this study isto evaluate the JEG-3 cells as a potential model system forplacental trophoblasts with regard to calcium transport.For this purpose, the cellular and biochemical propertiesand regulation of calcium handling by the JEG-3 cellshave been characterized. In addition, in view of evidencesuggesting that placental calcium transport may be undervitamin D regulation (Brunette, 1988; Brans et al. 1978;Danau et al. 1981; Halloran, 1989; Lester, 1986; Tanaka etal. 1979; Stumpf et al. 1983; van Bogaert, 1987), theresponse of JEG-3 cells to exogenous 1,25-dihydroxyvitamin D3, the active vitamin D metabolite, was alsocharacterized. The results reported here suggest that JEG-3 cells should serve as a valid and convenient in vitromodel system for studying the cellular mechanism andregulation of transplacental calcium transport by tropho-blasts.

Materials and methods

Cell cultureJEG-3 cells were obtained from the American Tissue TypeCulture and were grown in RPMI1640 (Cell-Gro) medium, 20 mMHepes, pH7.4, supplemented with fetal calf serum (10%) andpenicillin-streptomycin, and maintained at 37 °C in 5% COj.Optimal cellular attachment was obtained with Nunc or Costartissue culture plastic ware. In some experiments, the cultureswere supplemented with 1,25-dihydroxy vitamin D3 (BiomolResearch Lab., Inc.) at various concentrations for 24 h.

Placental CaBPDetection and quantitation. Immunodetection of CaBP in JEG-

3 cells was carried out using rabbit antibodies produced againstthe human placental CaBP (HCaBP) (Tuan, 1982, 1985). Cellswere washed in phosphate-buffered saline (PBS), and homogen-ized in a 20 mM Tris buffer (pH 7.4), centrifuged (31000g, 30 min),and the soluble extract was fractionated by SDS—polyacrylamidegel electrophoresis (12 % gel) (Laemmli and Favre, 1973). The gelwas then electroblotted onto nitrocellulose and immunoreactedwith anti-HCaBP followed by secondary antibodies conjugatedwith alkaline phosphatase, and then developed using a chromo-genic substrate system consisting of bromochloroindolyl phos-phate (BCIP) and nitroblue tetrazolium (NBT) (Ono and Tuan,1990). The immunoreactive HCaBP band was scanned densitome-trically using a Hoeffer scanner. The relative level of CaBP in dif-ferent JEG-3 samples was determined titrimetrically on the basisof signals generated from multiple serial dilutions of each sample.

Immunohistochemistry. Cells were fixed with 4 % paraformal-dehyde in PBS, rinsed with PBS, and incubated successively withanti-HCaBP antibodies, and fluorescein-conjugated goat anti-rabbit IgG antibodies (Tuan, 1986). After the final rinsing, theculture was mounted in PBS-glycerol and observed withepifluorescence and phase-contrast optics using an Olympus BH-2microscope. Photography was done using Kodak Tri-X film.

CaBP mRNADetection by blot hybridization. Total cellular RNA was

extracted from JEG-3 cells using the guanidine isothiocyanateprocedure (Chomczynski and Saachi, 1987). The integrity of theRNA preparations was routinely examined by means of in vitrocell-free translation in a rabbit reticulocyte lysate system(Promega), followed by SDS-polyacrylamide gel electrophoresis,to ascertain the presence of high-Afr (Sl60xl03) translatedproducts (Tuan and Kirwin, 1988). The RNA samples werefractionated by denaturing, formaldehyde-agarose (1%) gelelectrophoresis (Lehrach et al. 1979), and blotted onto GeneScreenfilter (New England Nuclear). The 0.7 kb CaBP cDNA insert wasexcised from pMCP (Tuan and Kirwin, 1988), 33P-labelled byrandom priming (Feinberg and Vogelstein, 1983), and used toprobe the RNA blot to detect CaBP mRNA (hybridization at 42 °Cin 50% formamide, 1 M NaCl, and 1% SDS). The Northern blotwas calibrated using RNA markers obtained from BRL. Inaddition, for CaBP mRNA quantitation, the RNA samples wereslot-blotted in serial dilution onto GeneScreen filter (NEN-DuPont) and probed similarly with labelled pMCP or /3-actincDNA (provided by Dr D. Cleveland). Hybridization signals werequantified by densitometric scanning of the autoradiograph.

Histolocalization by in situ hybridization. The non-radioactive,biotin-based procedure was described recently (Liebhaber et al.1989; McDonald and Tuan, 1989; Tuan et al. 1988). The cells wereplated on glass or plastic chamber slides (Miles or Nunc) that hadbeen coated with the cell culture bioadhesive, Adhera-Cell (GenexCorp.), fixed in modified Carnoy's fixative, dehydrated, digestedwith Proteinase K, denatured with formamide, and hybridizedwith pMCP, which had been biotin labelled by nick translationusing biotin-dUTP (BRL). Hybridization was detected usingstreptavidin conjugated with alkaline phosphatase, followed bychromogenic histochemistry using BCIP and NBT. Controlsincluded omission of probe or the use of irrelevant DNA as probe.

Placental Ca2+-activated ATPaseDetection and enzyme assay. JEG-3 cells were extracted in a

Tris buffer (pH7.4) containing 1% Triton X-100 (Tuan andBigioni, 1990; Tuan and Knowles, 1984; Tuan and Kushner,1987). Total Ca2+-ATPase enzyme activity, of both plasmamembrane and intracellular orgin, in the TritonX-100-solubilized JEG-3 extract was determined using themolybdate-Malachite Green assay as described previously (Tuanand Knowles, 1984; Tuan and Kushner, 1987), and expressed asnmol phosphate released min"1.

Histochemistry. Ca2+-ATPase activity was also detected histo-chemically using two previously described procedures (Tuan andBigioni, 1990; Tuan and Knowles, 1984; Tuan and Kushner,

334 R. S. Tuan et al.

1987). The first procedure involved electrophoretic fractionationof the solubilized cell extract on a non-denaturing Triton X-100polyacrylamide gel, which was then incubated with ATP in thepresence of PbCla, followed by Na2S to detect the precipitated Pb.Enzyme activity was visualized as a dark brown band on the gel.For the second, cytohistochemical procedure, cells were fixed with4 % paraformaldehyde in Hepes-Pipes buffer, pH 7.4, for 6 min atroom temperature, permeabilized with 0.01% Triton X-100 for15 s, rinsed with Tris-buffered saline, and incubated with ATP(3IBM) in the presence of PbCl2 for 30 min at 37 °C. The enzymereaction product was detected using Na2S to produce a brownishPbS precipitate, and the reacted cells were examined with bright-field and phase-contrast optics. Photography was done usingKodak Pan-X and Ektachrome films.

For all of the Ca2+-ATPase procedures described above, enzymeactivity was defined as the difference in ATPase activitymeasured with and without Ca2+. Thus, all controls includedomission of Ca2+, or the addition of, or pre-treatment of the gelwith, EGTA. An additional control for cytohistochemistry alsoinvolved the omission of ATP.

Cellular calcium uptakeAfter thorough rinsing of the tissue culture dish with Hank'sBalanced Salt Solution (Ca2+, Mg2+-free HBSS), cells wereincubated with gentle agitation in HBSS containing 0.01 minCaCl2 and trace amount of '"Ca at 37 °C for various periods oftime. At the end of the incubation, the cells were rinsed in coldPBS (3 times), solubilized with 2 % SDS, and the radioactivityincorporated was determined by liquid scintillation counting inEcolume (ICN). In other experiments, calcium concentration andincubation temperature were varied. The effect of various agentswas tested by pre-incubating the cells for 30 min and thenmeasuring uptake in the presence of the respective agents.Uptake activities were expressed as pmol calcium min"1.

Protein determinationThe BCA reagent of Smith et al. (1985) was used to determineprotein concentrations according to the protocol provided by themanufacturer (Pierce Chemicals).

Cytosolic [Ca2+] determinationThis was measured by the Fura-2 method as reported previously(Akins et al. 1988; Akins and Tuan, 1989; Grynkiewicz et al. 1985).Cells were rinsed with Ca2+, Mg2+-free Hank's Balanced SaltSolution (CMF-HBSS) to remove excess serum and calcium fromthe culture medium, and then treated with a solution of 5/JMFura-2 acetoxy methylester (Fura-2/AM) for 15 min at roomtemperature followed by 15 min at 37 °C. Cells were washed withCMF-HBSS, with 1 mM EGTA to remove unincorporated Fura-2/AM, and then mounted in a microscope cell chamber forobservation. Changes in cytosolic [Ca2+], as a function ofperfusion of the cells with 1-5 mM CaClj, were measured on thebasis of the Ca2+-dependent Fura-2 differential fluorescenceratios using the Deltascan calcium-imaging system of PhotonTechnology International (South Brunswick, NJ) connected to aLeitz Fluovert inverted microscope. In this procedure, cells loadedwith Fura-2 were initially viewed by phase-contrast microscopyto allow for optimal initial focusing and then alternately excitedwith either 350 or 380 nm (8 nm band pass) light. A single cell (orsubsection of a cell) was selected visually and extraneous imageswere blocked with a field aperture. Emitted light passed througha 495 nm long pass filter, and was detected by a photomultipliertube. The collected signals were corrected for backgroundfluorescence, and analyzed using the Deltascan System software.The ratio, #350/330, corresponded to cytosolic [Ca2+], and could bedetermined on the fly during the experiment. All determinationswere made using cells that yielded stable baseline fluorescencesignals at each of the excitation wavelengths, indicating that nodye leak had occurred.

The Fura-2 ratiometric signal was calibrated in vitro usinglOOmM KC1, lmM MgCl2, 10 mw EGTA, 5 mM Fura-2 and 10 mMPipes, pH7.0, with CaCl2 added to set the free [Ca2+]. Anempirical calibration curve was generated, and all experimentalR values were converted into cytosolic [Ca2+] values accordingly.

Results

JEG-3 cells in cultureCultured JEG-3 cells grew as large, connecting clusters,with a well-defined border surrounding the cellular mass(Fig. 1). During the early phase of culture, the generallycircular clusters (Fig. 1A and B) showed occasionalfibroblast-like cellular extensions when expansion orspreading was apparent. The cells appeared mostlysyncytial in the cluster, with a prominent nucleolarapparatus as well as various cytoplasmic particles(Fig. 1C); in addition, clear vacuole-like structures wereoften observed along the cellular cytoplasmic cortex.Because of their syncytial nature, JEG-3 cells wereroutinely passaged either by simply dividing scraped cellsinto new cultures or lifting them off the dish with a non-enzymatic cell dissociation solution (Specialty Media Inc.).Generally, cell scraping produced a culture of initialclusters containing about 30 cells each, whereas moreuniformly sized aggregates were usually obtained usingthe cell dissociation solution. With vigorous scraping,single cells could sometimes be released. Even duringlong-term culture, JEG-3 cells tended to remain as largelysyncytial aggregates; however, they did not exhibit anycontact inhibition, since at very high densities duringlong-term culture, the aggregates eventually grew on topof one another, giving rise to three-dimensional massesthat were clearly visible to the naked eye.

Expression of placental CaBPSince the placental CaBP was found to be specific forplacental membranes, it was of great interest to ascertainwhether it was also expressed by the JEG-3 cells,particularly if they were to be considered as potentialcandidate cells for the study of trophoblast calciumtransport. The expression of CaBP was analyzed at boththe protein and the mRNA levels.

CaBP. The presence of CaBP in JEG-3 cells wasdemonstrated using antibodies specific for the humanplacental CaBP (Tuan, 1982, 1985). Thus, both immuno-blotting (Fig. 2) and immunohistochemistry (Fig. 3) re-vealed the presence of the CaBP. As shown in Fig. 2, asingle high-M,. (~75xlO3) immunoreactive protein bandwas observed on the immunoblot, and corresponded to thatfound in the human term placenta. Immunohisto-chemistry (Fig. 3) revealed that the CaBP was associatedwith the cytoplasmic areas of the JEG-3 cell (Fig. 3A andB). Immunospecificity was demonstrated by the lack ofstaining in controls where antibodies to CaBP wereomitted (Fig. 3C and D). Interestingly, not all cells foundwithin an island stained positively for CaBP; distinct cellsshowing a total absence of immunostaining were observed.This finding strongly suggested that the cell populationwas heterogeneous with respect to CaBP expression, andfurther supported the notion that the cell islands were notcomplete syncytia.

CaBP mRNA. The expression of CaBP by JEG-3 cellswas also detected by RNA analysis involving blothybridization and in situ hybridization. The NorthernRNA blot (Fig. 4) clearly indicated the presence of a2.95 kb RNA hybridizing to the CaBP cDNA probe in bothplacental and JEG-3 total RNA isolates. When 0-actinmRNA hybridization signal was used as an internalstandard, JEG-3 cells were found to have a 1.7-fold higherrelative CaBP mRNA level than the placenta. In situhybridization of JEG-3 cells, as shown in Fig. 5, also

Trophoblast calcium uptake 335

STD JEG-3 JEG-3

Fig. 1. Morphology of JEG-3 cells in culture. (A) Lowmagnification of hematoxylin-stained cells (bright-field optics).JEG-3 cells appeared as large multinucleated, syncytial clumps(nuclei indicated by arrowheads). (B) Low magnification withphase-contrast optics. (C) Higher magnification (phase-contrast)showing prominent nuclei (n). Bar, 100 /an (A,B) or 10 /an (C).

2 0 5 -

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66-

45-

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Fig. 2. Detection of CaBP in JEG-3 cell extract byimmunoblotting. (A) Coomassie Blue-stained gel showing Afrstandards (xlO~3) and protein bands in two separate extractsof JEG-3 cells. Protein load was 16 fig in each sample lane.(B) Immuno-(alkaline phosphatase) staining of nitrocelluloseblot of gel shown in A. Antibodies to CaBP detected a singleprotein band of ~75xlO3 (arrowheads), which was similar tohuman term placental CaBP (Tuan, 1982).

revealed distinct cells with positive signals for the CaBP.Interestingly, as described above for immunohisto-chemistry of the CaBP (Fig. 3), the cDNA-mRNAhybridization signals were also heterogenously distrib-uted within the cell islands, since positive cells were foundmingled with negative cells.

Expression of placental Co2* -ATPaseThe analysis of the placental Ca2+-ATPase was based onenzyme activity in solubilized JEG-3 cell extract as well asin whole cells in situ.

Cell extract. A Triton X-100-solubilized extract of theJEG-3 cells exhibited significant Ca2+-activated ATPaseactivity, at a specific activity level of 13nmolPimin mg"1, which was slightly lower than thatreported for the human term placenta (Tuan and Kushner,1987). The membranous nature of the enzyme activity wasindicated as the activity was undetectable in non-detergent extracted cells. The enzyme activity of the JEG-3 cells was sensitive to a number of pharmacochemicalagents (Fig. 6A), similar to that of the human placenta(Tuan and Kushner, 1987). Further evidence supportingthe identity between the two enzymes was observed uponelectrophoretic fractionation of the activities (Fig. 6B).Thus, histochemical detection of enzyme activity on a non-denaturing electrophoretic gel showed that JEG-3 cellsand placental microsomes contained an activity band withidentical electrophoretic mobility, which corresponded tothat previously identified as associated with trophoblasts(Tuan and Kushner, 1987).

Enzyme cytohistochemistry. Ca2+-ATPase activity wasalso detected histochemically in paraformaldehyde-fixedJEG-3 cells (Fig. 7A,B). The staining was most intense inthe cellular periphery and in general appeared to be

336 R. S. Tuan et al.

Fig. 3. Immunohistochemical localization of CaBP in JEG-3 cells. Paraformaldehyde-fixed cells were immunostained with anti-CaBP antibodies as described in Materials and methods. (A,B) Immunostaining for CaBP (arrowheads in A, arrows in B) was seenclearly associated with a group of syncytial cells on the left of the micrograph, whereas other syncytial cells located at the rightand bottom (open arrows) were negative. A. Phase-contrast optics; B, epifluorescence optics. (C,D) Control with the omission ofantibodies to CaBP, showing the specificity of the immunostaining in A and B. C. Phase-contrast optics; D, epifluorescence optics.(E,F) CaBP immunostaining of JEG-3 cells treated in culture with (F) or without (E) 0.5 nM 1,25-dihydroxy vitamin D3 for 24 h.Epifluorescence optics for both E and F. Note the significantly elevated staining signal in the treated culture. Bar, 10 fan.Magnification identical for A-D and E-F, respectively.

Trophoblast calcium uptake 337

28S —

2.95 kb

18S —

Fig. 4. Detection of CaBP mRNA in JEG-3 cells by NorthernRNA blotting. Autoradiograms obtained after hybridizationwith CaBP cDNA probe (A), and hybridization of the same blotwith /3-actin cDNA probe (B). Lane 1, term placenta totalRNA; lane 2, JEG-3 total RNA. Each lane containedapproximately 10 j/g of RNA. The blot was calibrated with sizemarkers (kb=103 bases) as indicated; the electrophoreticmobilities of rRNAs are also indicated. A 2.96 kb CaBP mRNAband (arrow) was detected in both term placenta and JEG-3,whereas a 2.3 kb band was seen with the ^3-actin probe.

associated with areas of high cell density. Again, areaslacking any staining were also observed within a givenisland of cells, consistent with the cellular heterogeneitywith respect to CaBP expression. The enzyme specificityfor Ca2+ and ATP was also demonstrated in the histo-chemical reaction (Fig. 7C-K).

Calcium uptake activity of JEG-3 cellsJEG-3 cells exhibited active uptake of extracellularcalcium under the experimental conditions described inMaterials and methods. The characteristics of JEG-3calcium uptake are shown in Fig. 8A and B. Thus, calciumuptake was temperature-dependent and exhibited near-linear kinetics at 37°C for at least 7-8min (Fig. 8A). Theuptake activity also increased proportionally with me-dium calcium concentration (Fig. 8B). Calcium uptakewas dependent on the viability and membrane integrity ofthe JEG-3 cells, as paraformaldehyde fixation and digito-nin treatment both significantly reduced calcium uptakeactivity (data not shown). In addition, JEG-3 calciumuptake was sensitive to a number of pharmacochemicalagents. As shown in Fig. 8C, agents that were previouslyfound to inhibit the Ca2+-ATPase activity (Fig. 6A), inparticular quercetin and phenothiazin also effectivelyreduced calcium uptake by the JEG-3 cells. However,erythrosin B, which strongly inhibited Ca2+-ATPaseactivity (Fig. 6A), was ineffective on cellular calciumuptake (Fig. 8C), possibly due to membrane impermeab-ility to the drug. Overall, the similarity between thepharmacochemical sensitivity of the calcium uptake and

Ca2+-ATPase enzyme activities of the JEG-3 cells stronglysuggested that these two activities might be related.

To gain insight into the process of calcium handling bythe JEG-3 cells during active calcium uptake, cytosolic[Ca2+] was analyzed by Fura-2 microfluorometry. As

Ethacrynic acid

Quercetin

Phenothiazin

20 40 60 80% Enzyme activity

P J J P

Fig. 6. Characterization of Ca2+-ATPase activity of JEG-3cells. (A) Effect of various pharmacochemical agents; and (B)electrophoretic mobility on denaturing gel. In A, aolubilizedJEG-3 extract was first incubated with the respectivepharmacochemical agents at the indicated concentrations for10-15 min and then assayed in the presence of the agents atthe same concentration. All activities are expressed as apercentage of the control in the absence of any agents. Valuesrepresent the mean of 2-3 experiments. All agentssignificantly inhibited enzyme activity (P^0.06). In B,solubilized extracts of JEG-3 membranes and term placentalmicrosomes, prepared as described by Tuan and Kushner(1987), were subjected to Triton X-100 nondenaturrngpolyacrylamide (8 %) gel electrophoresis and stainedhistochemically for Ca2+-ATPase activity as described inMaterials and methods. Lane J, JEG-3; lane P, placenta. Leftpanel, histochemical reaction in the presence of 1 mM CaCl2;middle panel, in the absence of CaCl2; and right panel,Coomassie Blue protein staining profile. Protein load per lane:JEG-3, 0.55//g for histochemistry, 2.8//g for Coomassie Bluestaining; placental microsomes, 0.18 and 0.39 ng, respectively.Note the identical Ca2+-ATPase activity band seen in bothJEG-3 and placenta (arrowheads).

338 R. S. Tuan et al.

V•A

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Fig. 5. Localization of CaBP mRNA in JEG-3 cells by in situ hybridization. Biotinylated pMCP was used as the probe, and waslocalized after hybridization by means of alkaline phoaphatase histochemistry as described in Materials and methods. (A) Phase-contrast optics; (B) Nomarski differential interference contrast optics. Positive hybridization was seen as purple stain associatedwith the cell body. Note the localization of CaBP mRNA transcripts within several syncytical structures (arrows), and the absenceof signal in other cells (open arrows). Bar, 10 fan.

Fig. 7. Cytohistochemical localization of Ca2+-ATPase in JEG-3 cells. This was carried out as described in Materials and methods.A,B- The brownish-colored reaction product is shown in color (phase-contrast optics; A, before reaction; B, after reaction); seelocalization to the periphery of cells in high density areas (arrows). Negative cells were also seen (open arrows). (C-K) Black-and-white micrographs of enzyme histochemistry under various incubation conditions. (C-E) +Ca, +ATP; (F-H) -Ca, +ATP; and(I-K) +Ca, +AMP. Paraformaldehyde-fixed cells were first observed with phase-contrast (C,F,I) and bright-field (D,G,J) optics afterCa2+-ATPase reaction but prior to the addition of Na2S. The reaction product was then visualized as PbS precipitates and observedby bright-field optics (E,H»K). Positive reaction was only seen in the presence of both calcium and ATP (E); both positive cells(arrowheads) and negative cells (open arrow) were evident. Bar, 10/on.

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Fig. 8. Calcium uptake by JEG-3 cells. Calcium uptake wasmeasured as described in Materials and methods and expressedas pmolmg"1 protein. (A) Kinetics and temperaturedependence. (Inset: net calcium uptake at room temperatureand at 37°C after correcting for non-specific adsorption at 4°C.)The results were obtained from a typical experiment withtriplicates for each time point. (B) [Ca2+] dependence. Calciumuptake was measured in uptake buffers supplemented withCaClj at the indicated concentrations. All values are expressedas a percentage of that at 10 mM CaCl2. The data representresults (mean±s.D.) from three separate experiments, eachusing triplicates and covering various ranges of [Ca2+].(C) Sensitivity to various pharmacochemical agents. JEG-3cells were pre-incubated for 30 min with 10 \OA of each of theagents indicated and then assayed for calcium uptake at 37 °Cunder the same conditions. All values are the means of 2-3experiments and are expressed as a percentage of the controlvalue. Statistically significant difference (P<0.05) from thecontrol is denoted by an asterisk.

shown in Fig. 9, washed JEG-3 cells exposed to externalcalcium (5 mM) showed an immediate, rapid, and transientrise in cytosolic [Ca2+], preceding by a considerable timeinterval the actual cellular uptake of ^Ca (Fig. 8A). Infact, cytosolic [Ca2+] rapidly decayed to a plateau onlyslightly higher than the starting value. In addition, 0.5 nM1,25-dihydroxy vitamin D3 treatment, which greatlyenhanced calcium uptake (see below), did not significantlyalter the cytosolic [Ca2+] profile (Fig. 9B). From thiskinetic analysis, it appeared unlikely that the calciumuptake activity of JEG-3 cells could be accounted forsimply by a rise in cytosolic free [Ca2+]; an alternativecalcium sequestration mechanism was thus necessary.

ffe

50100 150

Time (s)

Fig. 9. Kinetic changes in cytosolic [Ca2+] during calciumuptake by JEG-3 cells. Cytosolic [Ca2+] was estimated byFura-2 microfluorimetry and expressed here as the ratio of theexcitation intensity at 351 nm to that at 380 ran, as describedin Materials and methods. JEG-3 cells were pre-incubated with1 mM EGTA and then exposed to CaCl2. [Ca2 +] was calculatedusing the formula of Grynkiewicz et al. (1985). (A) ControlJEG-3 cells, perfused with 5 mM CaCl2; (B) JEG-3 cells treatedwith 0.5 nM 1,25-dihydroxy vitamin D 3 for 24 h (see legend toFig. 10), perfused with 2 mM CaClj. The profiles shown hereare representative of those observed in typical experiments,where baseline [Ca2 +]=15-20 nM, peak [Ca2+] = ~600nM, andplateau [Ca2 +]=190-200 nM. Arrowheads indicate beginning ofperfusion.

Vitamin D regulation of calcium uptake and CaBPexpression by JEG-3 cellsTo investigate the effect of 1,25-dihydroxy vitamin D3,JEG-3 cells were treated with the hormone at variousdoses for 24 h and then analyzed for their calcium uptakeactivity and level of CaBP expression. As shown inFig. 10A, 1,25-dihydroxy vitamin D3 treatment signifi-cantly stimulated JEG-3 calcium uptake in a dose-dependent manner, the stimulation being maximal, 200 %,at 0.5 nM. Interestingly, the enhancement of cellularcalcium uptake was accompanied by a concomitantincrease in the steady-state level of the CaBP, asdetermined by quantitative immunoblotting. The resultsin Fig. 10B, based on densitometry of the immunoreactiveCaBP band on a Western blot, clearly indicated that 1,25-dihydroxy vitamin D3 treatment stimulated CaBP byapproximately twofold, compared to control. The stimu-

Trophoblast calcium uptake 339

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+ Vitamin D

0 10 20 30Protein (ue)

Fig. 10. Effect of 1,25-dihydroxy vitamin D3 on JEG-3 cells.(A) Calcium uptake activity. JEG-3 cells were treated with1,25-dihydroxy vitamin D3 for 24 h and then assayed forcalcium uptake as described in Materials and Methods.Significant stimulation was seen at 0.5 HM. Results are themeans of 3 separate experiments. Statistically significantdifference CP<0.05) from the control was observed at 0.5 and0.75 nM of 1,25-dihydroxy vitamin D3. (B) CaBP level.Immunoquantitation of CaBP was carried out by Western blotanalysis as described in Materials and methods. The signals, inarbitrary optical units, were obtained by densitometricscanning of the CaBP band on the blot using serial dilutions ofthe protein load. An approximately 2-fold increase in CaBP,based on the slope of the tdtration curve, was seen in JEG-3cells treated with 0.5 nM 1,25-dihydroxy vitamin D3 for 24 h.

lation of CaBP expression was also evident when JEG-3cells were immunostained with antibodies to CaBP; thus,cells treated with 1,25-dihydroxy vitamin D3 (Fig. 3F)exhibited significantly more intense staining than theuntreated control (Fig. 3E). On the other hand, quantitat-ive analysis of CaBP mRNA by serial RNA slot-blothybridization with radiolabelled pMCP, using /3-actinmRNA as an internal standard, did not reveal aconcomitant increase in the steady-state level of CaBPmRNA in the JEG-3 cells treated with 1,25-dihydroxyvitamin D3 (data not shown). Finally, the level of Ca2+-activated ATPase also remained unchanged in JEG-3 cellsafter 1,25-dihydroxy vitamin D3 treatment (data notshown).

Discussion

We have characterized the human choriocarcinoma cellline, JEG-3, as a candidate in vitro system for the study ofplacental calcium transport. The results reported herestrongly indicate that the JEG-3 cells mimic placental

trophoblasts with respect to many of the characteristicsand biochemical activities associated with calcium trans-port. These include the expression of two previouslyidentified marker molecules, a high-Mr CaBP and a Ca2+-activated ATPase, temperature- and substrate-dependentand kinetically linear calcium uptake, and responsivenessto 1,25-dihydroxy vitamin D3. Taken together with thesyncytial morphology of the JEG-3 cells, which resemblesthat of the syncytiotrophoblasts, these findings supportthe validity of these cells as an in vitro model for studyingplacental calcium transport.

The important function of the placenta as the sole tissueresponsible for nutrient translocation from the maternalto fetal circulation depends on the developmental differen-tiation of the trophoblasts into a specialized transportingepithelium (Dearden and Ockleford, 1983; Loke andWhyte, 1983). Thus, the syncytiotrophoblasts constitute athin, tight epithelial interface between the maternal andfetal circulations. Detailed understanding of the mechan-ism of transport therefore requires studying the tropho-blasts as an isolated cellular epithelium. Ideally, thisshould be a denned cell line that may be propagatedreproducibly and also continuously express propertiescharacteristic of the trophoblasts. The findings reportedhere are thus significant, since they establish the JEG-3cells, a stable cell line, as an in vitro experimental model ofplacental calcium transport.

The biochemical activities of the JEG-3 cells mimicthose of the term placental trophoblasts, with respect tothe CaBP and Ca*+-ATPase, both of which appear to beidentical to their placental counterparts. Thus, the JEG-3CaBP is immunoreactive with anti-human CaBP anti-bodies and has identical MT and subcellular distributionwith human placental CaBP; furthermore, the electro-phoretic mobility and pharmacochemical sensitivity of thetwo Ca2+-ATPase activities are also similar. (Note: totalcellular Ca2+-ATPase activities are analyzed, and thusboth plasma membrane and intracellular activities arecompared.) In addition, previous studies have also firmlyestablished that the endocrinological (Kohler and Bridson,1971; Patillo and Gey, 1968) and biochemical (Bahn et al.1981; Hamilton et al. 1979) properties of the JEG-3 cellsare similar to those of the placental syncytiotrophoblasts.

In this study we have characterized calcium uptake byJEG-3 cells cultured on tissue culture plastic. Thesubstrate and temperature dependence, linear kineticsand membrane integrity requirement of the uptakefunction are all consistent with an energy-requiring,cellular uptake process. Interestingly, uptake activity isstimulated by 1,25-dihydroxy vitamin D3 treatment of theJEG-3 cells, which also significantly increases the level ofthe cytosolic CaBP, but not the Ca2+-ATPase. It should benoted that calcium uptake as measured here is a net resultof influx and subsequent efflux of extracellular ^Ca.Consequently, it is reasonable to speculate that the effectof 1,25-dihydroxy vitamin D3 may be, first, the enhancedexpression of the cytosolic CaBP, which then acts toincrease retention of the influxed 4°Ca. Thus, the high-AfrCaBP of the placental trophoblasts may function in amanner similar to that of the low-Mr calcium-bindingprotein, calbindin-9K. Calbindin, first discovered as thevitamin D-dependent calcium-binding protein of theintestinal mucosa (Kallfelz et al. 1967; Marche et al. 1977),is present in the placenta (Bruns et al. 1978; Marche et al.1978) and appears to increase in level as a function ofgestation (Delorme et al. 1979). Various studies havesuggested that calbindin may act as a cytosolic calcium

340 R. S. Tuan et al.

sink to facilitate transepithelial calcium transport (Cara-foli, 1987; Wasserman and Fullmer, 1983), particularly inthe intestinal mucosa, although its exact functional roleremains to be established. It is thus noteworthy that in theJEG-3 cells, total cytosolic [Ca2+] did not rise concomi-tantly with calcium uptake, implicating a possible role forthe CaBP as an intracellular calcium sequestrator. Anumber of recent studies have also implicated calbindin asfunctionally involved in placental transport; in addition,another calcium-binding protein, oncomodulin (Brewerand MacManus, 1985), first identified in transformed cells,has also been identified in the placenta. How all theseproteins may participate in calcium transport, or possiblyother metabolic functions of the placenta, remains to beelucidated. Nevertheless, the regulatory role of 1,25-dihydroxy vitamin D3 in placental calcium transport isstrongly suggested, since both the high-Mr CaBP studiedhere and calbindin-9K are vitamin D-dependent. It shouldbe noted that, unlike calbindin-9K (Mathieu et al. 1989),the vitamin D-stimulated increase in the level of CaBPappears to be unaccompanied by a concomitant increase inmRNA. The exact mechanism of vitamin D stimulation ofCaBP expression therefore remains to be established.

Finally, the study of transepithelial transport in vitroideally requires the use of a tight, polarized cellularepithelial sheet that will mimic the vectorial translocationof solutes (Aimers and Stirling, 1984; Rodriguez-Boulanand Nelson, 1989; Sabatini et al. 1983; Simons and Fuller,1985), such as the widely studied MDCK cell line. Asdescribed earlier (Fig. 1), the JEG-3 cells will develop inlong-term culture into a cellular sheet consisting ofoverlapping cell clusters. Although these cell islands areheterogeneous with respect to the expression of the CaBPand Ca2+-ATPase, the fact that they form a cell sheet is adistinct advantage in the study of transepithelial trans-port. Experiments are underway to examine the tightnessand the polarity of the JEG-3 cell sheet. Initial obser-vations on JEG-3 cells cultured on permeable membranesubstrata suggest that a tight epithelium is indeed formedin vitro.

In summary, the studies presented here have provided afirm basis for the application of the human choriocarci-noma cell line, JEG-3, as an in vitro cellular model for thestudy of calcium transport by placental trophoblasts. Inparticular, the phenotypic stability and the hormoneresponsiveness of these cells should greatly facilitate theunderstanding of the mechanism and regulation oftrophoblast calcium transport.

This work was supported in part by grants from the NIH (HD15822, HD 21365), March of Dimes Birth Defects Foundation(1-1146), and the U.S. Department of Agriculture(88-37200-3746). The technical assistance of Ken Shepley in thein situ hybridization experiment is also acknowledged.

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{Received 29 October 1990 - Accepted 18 December 1990)

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