Tài liệu Báo cáo khoa học: Trophoblast-like human choriocarcinoma cells serve as a suitable in vitro model for selective cholesteryl ester uptake from high density lipoproteins pdf

Eur J Biochem 270, 451–462 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03394.x Trophoblast-like human choriocarcinoma cells serve as a suitable in vitro model for selective cholesteryl ester uptake from high density lipoproteins Christian Wadsack1, Andelko Hrzenjak1, Astrid Hammer2, Birgit Hirschmugl1, Sanja Levak-Frank1, Gernot Desoye3, Wolfgang Sattler1 and Ernst Malle1 Institute of Medical Biochemistry and Molecular Biology, 2Institute of Histology and Embryology, 3Clinic of Obstetrics and Gynecology, Karl-Franzens University Graz, Austria As human choriocarcinoma cells display many of the biochemical and morphological characteristics reported for in utero invasive trophoblast cells we have studied cholesterol supply from high density lipoproteins (HDL) to these cells Binding properties of 125I-labeled HDL subclass (HDL3) at °C were similar for BeWo, JAr, and Jeg3 choriocarcinoma cell lines while degradation rates at 37 °C were highest for BeWo Calculating the selective cholesteryl ester (CE)-uptake as the difference between specific cell association of [3H]CE-labeled HDL3 and holoparticle association of 125I-labeled HDL3 revealed that in BeWo cells, the selective CE-uptake was slightly lower than holoparticle association However, the pronounced capacity for specific cell association of [3H]CE-HDL3 and selective [3H]CE-uptake in excess of HDL3–holoparticle association, and cAMP–mediated enhanced cell association of [3H]CE-HDL3 in JAr and Jeg3 suggested the scavenger receptor class B, type I (SR-BI) to be responsible for this pathway Abundant expression of SR-BI (but not SR-BII, a splice variant of SR-BI) could be observed in JAr and Jeg3 but not in BeWo cells using RT-PCR, Northern and Western blot analysis, and immunocytochemical technique Adenovirus-mediated overexpression of SR-BI in all three choriocarcinoma cell lines resulted in an enhanced capacity for cell association of [3H]CE-HDL3 (20-fold in BeWo; fivefold in JAr and Jeg3) The fact that exogenous HDL3 remarkably increases proliferation in JAr and Jeg3 supports the notion that selective CE-uptake and subsequent intracellular generation of cholesterol is coupled to cellular growth From our findings we propose that JAr and Jeg3 cells serve as a suitable in vitro model to study selective CE-supply to human placental cells Mammalian fetal nutrition during development is wholly dependent on the transport of nutrients by the placenta Cholesterol is not only a major component of cell membranes but is also necessary for the maintenance of fetal growth and serves as the initial substrate for steroid hormone synthesis [1] There are two sources of cholesterol in the fetus, as in any other tissues The first source is endogenous and constitutes cholesterol synthesized within the fetus The second source of fetal cholesterol is exogenous [2,3] Exogenous cholesterol could be derived from cholesterol synthesized in the yolk sac and placenta or by transport of lipoprotein-associated cholesterol across the placenta from the maternal circulation [4–8] The fact that the placenta binds and internalizes maternal lipoproteins both in vivo and in vitro [8] suggests the existence of functionally intact lipoprotein receptors [2–11] The presence of active lipoprotein receptors in the yolk sac, the placenta and placenta-derived cells support internalization of cholesterol-rich maternal lipoproteins [8] In addition to low-density lipoprotein (LDL) that are taken up by the classical receptor-mediated endocytosis pathway (the LDLor apolipoprotein B/E-receptor), the high clearance rate of high-density lipoprotein (HDL) in rodents suggested specific HDL receptors [8] contributing to sterol metabolism in fetal tissues Among these gp280 preferentially mediates HDLholoparticle-uptake [9] while scavenger receptor class, B, type I (SR-BI), the predominant receptor mediating selective uptake of cholesteryl esters (CEs) without concomitant holoparticle-uptake [12,13], has been identified in steroidogenic tissue and the liver [12] as well as in human [14] and rodent placental tissues [15,16] Immunohistochemistry revealed strong induction of SR-BI expression in murine giant trophoblast cells that surrounded the developing embryo [16] The fact that murine SR-BI (mSR-BI) is Correspondence: E Malle, Karl-Franzens University Graz, Institute of Medical Biochemistry and Molecular Biology, Harrachgasse 21, A)8010 Graz, Austria; Fax: +43 316 380 9615; Tel.: +43 316 380 4208; E-mail: ernst.malle@kfunigraz.ac.at Abbreviations: [3H]CE, [1,2,6,7–3H]-cholesteryl palmitate; CE(s), cholesteryl ester(s); CHO, Chinese hamster ovary; 8-CPTcAMP, 8-(4-chlorophenylthio)adenosine 3¢:5¢-cyclic monophosphate; DMEM, Dulbecco’s minimum essential medium; FBS, fetal bovine serum; HBSS, Hank’s balanced salt solution; HDL3, high density lipoprotein subclass 3; LDL, low density lipoprotein; LPDS, lipoprotein-deficient serum; mSR-BI, murine scavenger receptor class B, type I; TCA, trichloroacetic acid (Received 22 July 2002, revised 10 October 2002, accepted 26 November 2002) Keywords: BeWo; JAr; Jeg3; placenta; SR-BI Ó FEBS 2003 452 C Wadsack et al (Eur J Biochem 270) expressed on the side of the tissue that faces the maternal blood expression supports the notion that this protein could act as a candidate receptor for supplying cholesterol to developing embryonic tissues for placental steroid biosynthesis The process of binding and uptake of maternal lipoproteins is carried out by placental trophoblast [17], which lines the chorionic villi and represents the epithelial layer separating the maternal and fetal circulation [18] In the human placenta chorionic villi, a layer of syncytiotrophoblasts lies over the cytotrophoblasts, and surrounds the internal mesoderm and fetal capillaries [18] Trophoblasts may also give rise to choriocarcinoma which are malignant tumors of epithelial origin but have been shown to display characteristics of invasive trophoblasts [19] Choricarcinoma cells are morphologically similar to their cell of origin, the trophoblast of the normal first trimester placenta and may serve as a valid and convenient in vitro model system for studying the cellular activities and regulation of transplacental transport and uptake mechanisms To date, only limited information on binding and holoparticleuptake of lipoproteins by choriocarcinoma cell lines is available [20,21] However, no studies have been performed to clarify whether choriocarcinoma cell lines may serve a suitable in vitro model for supplying cholesterol to placental cells via selective CE-uptake from HDL We therefore have studied holoparticle association of and selective CE-uptake from HDL by three human trophoblast-like choriocarcinoma cell lines, i.e BeWo, JAr, and Jeg3 The BeWo cell line which is heterogenous on several criteria [22], is comprised of cytotrophoblasts with no differentiation to syncytium [23] under non activated conditions [24] This is in contrast to primary cultures of term cytotrophoblasts which aggregate and form syncytia In contrast to BeWo and Jeg3 cell lines, JAr cells share many of the characteristics of early placental trophoblasts, such as synthesis of human chorionic gonadotropin and steroids [25], and the ability to differentiate into syncytiotrophoblastlike cells in vitro [19] Jeg3, derived originally from the BeWo [26], express abundant human chorionic gonadotropin and placental lactogen – hallmarks of trophoblasts [27] – and form large, multinucleated syncytia in culture [28] which resemble that of syncytiotrophoblasts in vivo Results from the present study demonstrate that the capacity of selective CE-uptake by all three choriocarcinoma cell lines is closely related to the expression level of SR-BI Only JAr and Jeg3 could serve as suitable in vitro models to study selective CE supply to human placental cells High level adenoviral-overexpression of SR-BI resulted in an enhanced and comparable capacity for selective CE-uptake by all three choriocarcinoma cell lines The lack of SR-BII protein suggested that this splice variant of SR-BI [29] does not contribute to selective CE-uptake by BeWo, JAr, and Jeg3 cells Experimental procedures Human lipoproteins LDL (d ẳ 1.0351.065 gặmL)1) and HDL subclass (HDL3, lacking apolipoprotein E, a ligand for the LDLreceptor, d ẳ 1.1251.21 gặmL)1) was prepared by discontinuous density ultracentrifugation from plasma obtained from normolipidemic blood donors [30] Purity of lipoprotein preparations was checked by SDS/PAGE The protein concentration was determined as described [31] using bovine serum albumin as a standard Oxidation of LDL (ox-LDL) was performed with 1.66 lM of Cu2+ in NaCl/Pi as described [32] Lipoprotein labeling procedures Single-labeling (a) Iodination of HDL3 with 125I-Na (NEN, Vienna, Austria) was performed using N-Br-succinimide as the coupling agent [33] This procedure resulted in specific activities between 300 and 500 d.p.m.Ỉng)1 protein with less than 3% lipid associated activity All 125I-HDL3preparations were monitored by SDS/PAGE to ensure that the preparations were free of radiolytic or oxidative damage (b) HDL3 was labeled with [cholesteryl-1,2,6,7–3H]-palmitate ([3H]CE, NEN) by cholesteryl ester transfer, proteincatalyzed transfer from donor liposomes exactly as described [34] Subsequently, the labeled HDL3-fractions were reisolated in a TLX120 bench-top ultracentrifuge in a TLA100.4 rotor (Beckman, Austria) The HDL3-band was aspirated and dialyzed against 10 mM NaCl/Pi, pH 7.4 Double-labeling Labeling of HDL3 with [cholesteryl1,2,6,7–3H]-oleate (NEN) was performed by incubation of the lipoprotein with palmityl oleyl phosphatidylcholine[3H]cholesteryl oleate vesicles exactly as described [35] Following separation from the vesicles by ultracentrifugation, the labeled lipoproteins were subsequently labeled with 125 I-Na as described above using N-Br-succinimide Cell culture studies Choriocarcinoma cells The BeWo cell line (ATCC, Manassas, VA, USA) was cultured in F12K (Kaighn’s Modification) Nutrient Mixture (Gibco BRL, Vienna, Austria) supplemented with 10% (v/v) heat inactivated fetal bovine serum (FBS, hyClone, Utah, USA) containing 100 mL)1 penicillin/streptomycin and mM L-glutamine at 37 °C under 5% CO2 The JAr and Jeg3 cell lines (ATCC) were maintained in Dulbecco’s minimum essential medium (DMEM, supplemented with 10% [v/v] FBS and 100 mL)1 penicillin/streptomycin) at 37 °C under 5% CO2 To assess influences of exogenous HDL3 on cell proliferation, the cells were seeded into 6-well plates at a starting density of 2.5 · 104 cells in mL 10% lipoproteindeficient serum (LPDS) medium for BeWo, · 104 cells for JAr and · 105 cells for Jeg3 cells, respectively After 24 h, media was removed, the cells were washed with Hank’s balanced salt solution (HBSS), trypsin-treated and counted with a cell counter and analyzer system (CASYÒ1, Scharfe, ă Reutlingen, Germany) Fresh medium containing lipoprotein-decient serum or lipoprotein-deficient serum plus 100 lgỈmL)1 HDL3 was added and the cells were incubated At the indicated time points the cells were washed twice with HBSS, trypsin-treated and counted Chinese hamster ovary (CHO) cells LdlA7 (clone 7, a LDL receptor-negative CHO cell line) which express minimal levels of SR-BI were cultured in HAM’s Ó FEBS 2003 Human choriocarcinoma cells and SR-BI (Eur J Biochem 270) 453 F12 medium containing 5% (v/v) FBS, mM glutamine, 50 mL)1 penicillin and 50 lgỈmL)1 streptomycin [36]; ldlA(mSR-BI) (ldlA7 stably transformed with mSR-BI) were maintained in medium containing 0.5 mgỈmL)1 G-418 Both cell lines were kindly provided by Dr M Krieger (MIT, Cambridge, MA, USA) Binding studies Binding studies of human HDL3 to choriocarcinoma cells were performed at °C in medium (F12K/DMEM without FBS) with increasing amounts of 125I-labeled HDL3 (1, 5, 10, 25, 50, and 100 lg proteinỈmL)1) in the absence (total binding) or presence of a 20-fold excess of unlabeled HDL3 (nonspecific binding) [37] Following this incubation the medium was aspirated and the cells were washed twice with HBSS containing 5% (v/w) bovine serum albumin followed by two washes in HBSS Cells were lysed with 0.3 M NaOH (1 mL, h at °C) to determine bound-radioactivity and cell protein in the lysate Protein measurement was performed as described [31] Specific binding (4 °C) was calculated as the difference between total and nonspecific binding [37] To determine cell-associated (the sum of binding and internalization) and degraded 125I-labeled HDL3-protein, the cells were incubated at 37 °C for h as described above with the same amounts of labeled and unlabeled HDL3 Subsequently, the medium was aspirated and the cells were rinsed as described above [34] Degradation of 125I-labeled HDL3 by choriocarcinoma cells was estimated by measuring the nontrichloroacetic acid-precipitable radioactivity in the medium after precipitation of free iodine with AgNO3 exactly as described [38] Briefly, 0.5 mL of medium was removed, mixed with 100 lL bovine serum albumin (30 mgỈmL)1) and mL trichloroacetic acid (3 M, °C) and left at °C for 30 Subsequently 250 lL of AgNO3 (0.7 M) was added, mixed, and the samples were centrifugated at 1500 · g at °C for 15 One milliliter of the supernatant was counted on a c-counter To determine cell association of tritiated-HDL3, the cells were incubated with [3H]CE-HDL3 at 37 °C for h as described above with the same amounts of labeled and unlabeled HDL3 [34] After removing the medium and washing the cells, cell association was estimated by measuring the radioactivity and protein content of the cell lysates, respectively Specific cell association was calculated as the difference between total and nonspecific cell association In a parallel set of experiments, the cell association of [3H]CE-HDL3 by choriocarcinoma cells was measured by increasing concentrations of unlabeled competitors (HDL3 and ox-LDL) as well as in the absence or presence of 300 lM 8-(4-Chlorophenylthio)adenosine 3¢,5¢-cylic monophosphate (8-CPTcAMP, Sigma, Vienna) particles only specific cell-associated radioactivity (the difference between total and nonspecific cell association of [3H]CE-HDL3) was counted To facilitate quantitative comparison of 125I-labeled and [3H]CE association, results are expressed in terms of HDL3-protein equivalents [39] calculated on the basis of the specific activity of the correspondingly labeled HDL preparation used that would be necessary to deliver the observed amount of tracer These calculations are performed to compare uptake of 125I- and H-tracers on the same basis In a parallel set of experiments, the selective CE-uptake from HDL3 was calculated from double-labeled HDL3 as the difference between [3H]cholesteryl oleate- and 125Ilabeled lipoprotein [35] The sum of specific degradation (measured as described above) and specific cell association of 125 I-labeled HDL3 reflects HDL3–holoparticle association SDS/PAGE and Western blotting Detergent extracts of solubilized membrane protein fractions [33] or total cell proteins of choriocarcinoma and CHO cells were separated on 8% SDS/PAGE Protein transfer to nitrocellulose membranes was performed electrophoretically at 150 mA, °C, 50 [34] Immunochemical detection of SR-BI and SR-BII (a splice variant of SR-BI lacking the C-terminal portion [29]) was performed with a sequence-specific rabbit anti-SR-BI-peptide (496–509) and anti-SR-BII peptide (491–506) IgG (dilutions : 1500, Abcam, Cambridge, UK) Immunoreactive bands were visualized with peroxidase-conjugated goat anti-rabbit IgG and ECL development [30] Reverse-transcriptase-polymerase chain reaction Total RNA from choriocarcinoma cell lines was isolated by using RNeasy kit (Qiagen, Vienna, Austria) Three micrograms of total RNA were treated with RQ1 RNase-free DNase I (Promega, Mannheim, Germany) for 15 at 37 °C and subsequently used as a template for first strand cDNA synthesis in a 30-lL reaction The PCR conditions applied were described in detail elsewhere [40] The following oligonucleotides were used: forward primer (A) 5¢-TCTACCCACCCAACGAAGGCT-3¢ (nucleotides 1007–1027), SR-BI reverse primer (B) 5¢-CCTGAATGGC CTCCTTATCCT-3¢ (1514–1534) and SR-BII reverse primer C-5¢-AGAAGCGGGGTGTAGGGACTGG-3¢ (1655–1676) (MWG Biotech, Ebersberg, Germany) By using the primers A and B, a 527-bp fragment was obtained By using the primers A and C, two fragments were obtained: a 669-bp-fragment for SR-BI and a 540-bp-fragment for SR-BII (129 bp shorter than SR-BI) All fragments were subcloned in TOPO-TA cloning vector and sequenced Selective CE-uptake Northern blot analysis To calculate the selective CE-uptake from HDL3 during the same experiment, choriocarcinoma cells were incubated with 125I-labeled and [3H]CE-HDL3 In case of 125I-labeled HDL3, the cell-associated and nontrichloroacetic acidprecipitable radioactivity in the medium was counted The sum of cell-associated and degraded 125I-labeled HDL3 reflects holoparticle association For the [3H]CE-HDL3- Total RNA was isolated from choriocarcinoma and human liver tissues (used as a positive control) by the RNA-easy kit (Qiagen) exactly as described [40] A 553-bp fragment, amplified by RT-PCR [forward primer 5¢-TCGCTCAT CAAGCAGCAGGT-3¢ (nucleotides 169–188), reverse primer 5¢-GCCCAGAGTCGGAGTTGTTG-3¢ (nucleotides 702–721)] was used as a probe [30] Ó FEBS 2003 454 C Wadsack et al (Eur J Biochem 270) Construction of recombinant human SR-BI adenovirus The adenoviral plasmid shuttle vector (pAvCvSv) and pJM17 vector were kindly supplied by L Chan (Baylor College of Medicine, Houston, Texas) Human SR-BI cDNA (kindly provided by H Hauser, ETH, Zurich, ă Switzerland) which was originally inserted into pcEXV-3 vector was partially restricted with EcoR I and the 2.5 kb band was eluted from the gel This band was subcloned into pBluescript using the EcoRI site, amplified, restricted with Kpn-I and this fragment was finally partially restricted with BamHI The plasmid shuttle vector was opened using Kpn-I and BglII, and Kpn-I/BamHI restricted SR-BI cDNA was inserted These modifications were necessary to enable insertion of SR-BI cDNA under the control of the CMV promoter in the plasmid shuttle vector Recombinant vector (pAvCvSv/human SR-BI) was used to transform E coli DH5-a competent cells in order to amplify the recombinant plasmid Positive clones were confirmed by restriction analysis and DNA-sequencing The resulting recombinant shuttle plasmid (5 lg) was cotransfected with lg supercoiled pJM17 into 293 cells by calcium-phosphate coprecipitation Two weeks after transfection, infectious recombinant adenoviral vector plaques were picked, propagated, and screened for SR-BI sequences by PCR Adenoviral vectors containing SR-BI were further amplified in 293 cells and the expression was determined by Western blotting analysis Large-scale production of high-titer recombinant adenovirus was performed as described [41] The virus was purified twice by caesium chloride density gradient centrifugation and dialyzed for 14 h at °C against a buffer containing 10 mM Tris/HCl, pH 7.5, mM MgCl2, 10% glycerol and stored at )70 °C Virus-titer was determined by plaque-assay on 293 cells and was found to be · 1010 pfmL)1 Control b-galactosidase (b-gal) and viruses were amplified and purified as described above Adenovirus infection of choriocarcinoma cells Choriocarcinoma cells were cultivated in 12-well culture dishes At a density of · 104 cells per cm2 they were rinsed once with NaCl/Pi and infected with recombinant adenoviruses (MOI ẳ 30 pfuặmL)1) in mL of infection media (DMEM medium containing 2% FBS, 50 mL)1 penicillin, and 50 lgỈmL)1 streptomycin) for h (37 °C, 5% CO2, humidified atmosphere) After removing infection-media, the cells were supplied with mL of S12K medium (containing 5% FBS, mM glutamine, 50 mL)1 penicillin and 50 lgỈmL)1 streptomycin) and the incubation was continued for 36 h without rocking Control cells were infected with b-gal virus as described for SR-BI infected cells The expression rate of SR-BI was determined by Northern and Western blot techniques as described above cultured on Laboratory-Tek chamber slides (Nunc, USA) After 24 h the cells were washed with HBSS, air dried (2 h at 22 °C), and acetone-fixed (5 min) [42] Fixed chamberslides were incubated for 30 with rabbit anti-SR-BI IgG (dilutions of : 1000, NB 400–104, Novus Biologicals, USA) followed by a 30-min incubation with cyanine3-labeled goat anti-rabbit IgG (dilution : 400, Jackson Dianova) NaCl/Pi was used for washing sections between different incubation steps Sections were mounted with Moviol (Calbiochem-Novabiochem, La Jolla, CA) and analyzed on a confocal laser scanning microscope (Leica TCS NT, Heidelberg, Germany) equipped with an argonkrypton laser Signals were detected with a double dichroitic beam splitter (488/568 nm), using a 590-nm long pass filter for cyanine-3 [42] Control experiments encompassed immunocytochemistry (a) without primary detection antibodies (b) with polyclonal nonimmune antibodies as primary antibodies (c) without secondary antibodies, or (d) using ldlA(mSR-BI) membrane protein fractions as competing antigen; the competitor (20-fold molar excess) was preincubated with the primary antibody for 20 before adding to the section Pictures were taken with an Axiophot microscope (Zeiss, Oberkochen, Germany) Results Binding of HDL to choriocarcinoma cells Specific binding of HDL3 to all three choriocarcinoma cell lines at °C was saturable at the highest lipoprotein concentrations (100 lg proteinỈmL)1) used Calculation of binding parameters yielded similar Kd and bmax values for all three cell lines (Table 1) Also the specific cell association of 125 I-labeled HDL3 (the sum of binding and internalization) measured at 37 °C was comparable for all three cell lines (Fig 1A–C) The specific degradation of 125I-labeled HDL3 determined in a parallel set of experiments was highest for BeWo and decreased in the following order: BeWo > JAr > Jeg3 Selective HDL-CE uptake by choriocarcinoma cells For BeWo cells, the specific cell association of [3H]CEHDL3 increased up to 1200 ng HDL3Ỉmg)1 cell protein (Fig 2A) and is approximately 1.8-fold higher than 125 I-labeled HDL3–holoparticle association (the sum of specific cell association and degradation of 125I-labeled Table Specific binding of 125I-labeled HDL to choriocarcinoma cell lines at °C Binding constants were calculated by non linear regression analysis (GRAPHPAD) Values are given as means ± SD of three independent experiments Immunofluorescence and confocal laser scanning microscopy Immunofluorescence was performed on choriocarcinoma cells (24 h in DMEM containing 10% FBS, 1% glutamine and 1% penicillin/streptomycin) and CHO cells (HAM’s F12 medium containing 5% (v/v) FBS, mM glutamine, 50 mL)1 penicillin and 50 lgỈmL)1 streptomycin) Kd (lg HDL3-proteinỈmL)1) BeWo JAr Jeg3 bmax (ng HDL3-proteinỈmg)1 cell protein) 41.9 ± 10.1 34.1 ± 3.9 29.7 ± 7.4 161.3 ± 17.3 145.9 ± 6.9 186.9 ± 21.2 Ó FEBS 2003 Human choriocarcinoma cells and SR-BI (Eur J Biochem 270) 455 Fig Specific cell association and degradation of 125I-labeled HDL by choriocarcinoma cells Following preincubation in F12K, [BeWo (A)], or DMEM [JAr (B) and Jeg3 cells (C) without FBS (12 h)], the cells were incubated for h (37 °C) in the presence of increasing amounts of 125 I-labeled HDL3 To determine specific cell association of 125I-labeled HDL3 (closed triangles), the cells were incubated in the absence (total cell association) or presence of a 20-fold excess of unlabeled HDL3 (non specific cell association) The cells were washed and lysed with 0.3 M NaOH to determine the cell-associated fraction (the sum of bound and internalized radioactivity) To determine specific degradation (closed circles – the difference between total and nonspecific degradation) the cells were incubated under the same conditions as described above After h the cellular supernatant was collected to determine the nontrichloroacetic acid-precipitable radioactivity as described in Materials and methods (ÔdegradedÕ) Results are given as means ± SD of three independent experiments HDL3) However, calculating the selective CE-uptake from HDL3 as the difference between both tracers revealed that this pathway is not a preferential routing to supply BeWo with CEs from HDL3 To confirm the low capacity of BeWo cells for selective CE-uptake, the HDL3-particle was double-labeled and values obtained for specific cell association of [3H]cholesteryl oleate- and 125I-labeled HDL3 were compared with those obtained by single labeling experiments Comparable results were obtained with both labeling techniques (data no shown) In contrast to BeWo, JAr cells showed a pronounced capacity for specific CE tracer uptake from HDL3 in excess of holoparticle association, exceeding holoparticle association by approximately fivefold (Fig 2B) In parallel, the selective CE–uptake exceeded holoparticle association by approx 4-fold (2703 vs 652 ng lipoproteinỈmg)1 cell protein) at the highest HDL3-concentrations used (Fig 2B) Although specific cell association of [3H]CE-HDL3 in Jeg3 cells (Fig 2C) was similar to BeWo (Fig 2A), the capacity for selective CE-uptake from HDL3 exceeded holoparticle association by approximately sixfold (1537 vs 267 ng lipoproteinỈmg)1 cell protein) in these cells Identification and characterization of SR-BI Based on the pronounced capacity of JAr and Jeg3 cells (Fig 2B,C) for selective uptake of HDL3-associated CEs it was reasonable to assume that this pathway is caused by high expression levels of SR-BI Using specific primers for SR-BI the corresponding 527 bp product (primer A and B, Fig 3A) and 669 bp product (primer A and C, not shown) was abundantly present in JAr and Jeg3 cells while only negligible amounts became apparent in BeWo cells In parallel (using primers A and C) a faint 540 bp band (indicative for SR-BII) was found in all three cells lines (not shown) Northern blotting experiments confirmed data obtained by RT-PCR (Fig 3B) Western blot experiments finally revealed high expression of SR-BI in JAr and Jeg3 cells (Fig 3C) In BeWo cells, marginal SR-BI expression was observed on RNA level but no SR-BI protein was detectable under the conditions described (Fig 3) Western blot experiments of detergent solubilized membrane protein fractions revealed no expression of SR-BII on all three choriocarcinoma cell lines (not shown) Using specific antibodies, pronounced staining for SR-BI could be observed primarily on the plasma membrane of JAr and Jeg3 cells and to a much lesser degree in the cytoplasma of both cell lines (Fig 4B,C) Omission of the primary antibody (data not shown) or replacement of primary antibodies with an IgG isotype control (data not shown) eliminated all staining Competition experiments of antiSR-BI IgG preabsorbed with SR-BI-enriched membrane protein fraction from ldlA(mSR-BI) cells at a molar ratio of : 20 prevented antibody binding, demonstrating that staining was specific for SR-BI The lack of SR-BI expression in BeWo cells (Fig 3C) could be confirmed by 456 C Wadsack et al (Eur J Biochem 270) Ó FEBS 2003 Fig Specific cell association of [3H]CE-HDL and selective CE-uptake from HDL by human choriocarcinoma cells Following preincubation in F12K [BeWo (A)], or DMEM [JAr (B) and Jeg3 cells (C) without FBS (12 h)], the cells were incubated for h with increasing amounts of [3H]CE-HDL3 at 37 °C To determine specific cell association of [3H]CE-HDL3 (closed circles) the cells were incubated in the absence (total association) or presence of a 20-fold excess (nonspecific association) of unlabeled HDL3 Subsequently, the cells were washed and lysed in 0.3 M NaOH to measure associated radioactivity The selective CE-uptake (closed triangles) was calculated as the difference between specific cell association of [3H]CE-HDL (closed circles) and 125I–labeled HDL holoparticle association (closed squares, the sum of specific cell association and degradation, Fig 2) Results are given as means ± SD of three independent experiments immunocytochemistry (Fig 4A) To further verify specificity of the primary antibody on choriocarcinoma cells, CHO cells were used as controls While faint staining was observed on ldlA7 cells (expressing minimal levels of SR-BI, Fig 4E), bright staining could be observed on ldlA(mSR-BI) cells (Fig 4D) SR-BI expressed on choriocarcionoma cells contributes to CE-uptake from HDL To further confirm that SR-BI accounts for the high rates of selective CE-uptake by JAr and Jeg3 cells, a series of competition experiments were performed While HDL3 and ox-LDL (a high affinity ligand for SR-BI [10]) were equally effective to compete for cell association of [3H]CE-HDL3 in BeWo cells, HDL3 lowered cell association of [3H]CEHDL3 by approx 25% in JAr and Jeg3 cells (Table 2) Further inhibition (up to 60%) was achieved by increasing the competitor concentration of unlabeled HDL3 to 500 lgỈmL)1 The addition of ox-LDL led to a pronounced inhibition in JAr and Jeg3 cells, findings in line with previous results [43] performed on liver cells that express high levels of SR-BI Next, cell association from [3H]CE-HDL3 was studied in cells preincubated with 8-CPTcAMP, a direct activator of SR-BI via cAMP-dependent protein kinase pathway [10,11] In line with findings shown in Fig 2, cell association of [3H]CE-HDL3 is lowest in BeWo compared with JAr and Jeg3 cells (Fig 5) Following 8-CPTcAMP treatment, cell association of [3H]CE-HDL3 was unaltered in BeWo but increased in JAr and Jeg3 cells These changes were paralleled by SR-BI mRNA levels (Fig 5) Finally, we tested whether transient overexpression of SR-BI would restore the ability of BeWo for cell association of [3H]CE-HDL3 Therefore, choriocarcinoma cell lines were transiently transfected with the human SR-BI gene and expression was followed by Northern blot (data not shown) and Western blot experiments (Fig 6) The SR-BI protein expressed was predominantly localized at the plasma membrane as determined by immunoblot analysis of plasma membrane preparations In order to test the functionality of the adenoviral SR-BI construct, the cells were incubated with [3H]CE–HDL3 and cell association of [3H]CE-HDL3 was measured While mocktransfection of all three choriocarcinoma cells did not change the capacity for cell association of [3H]CE-HDL3 compared with wild type cells, adenoviral overexpression increased the capacity for cell association of [3H]CE-HDL3 approximately fivefold (JAr and Jeg3) and 20-fold (BeWo), respectively From this set of experiments, we conclude that transfection of all three choriocarcinoma cell lines results in functionally active SR-BI protein, and that transfection in BeWo (a cell line with low SR-BI expression comparable to ldlA7 cells [44]) results in cell association of [3H]CE-HDL3 to an extent similar as shown with JAr and Jeg3 under the same conditions Ó FEBS 2003 Human choriocarcinoma cells and SR-BI (Eur J Biochem 270) 457 cellular cholesterol content of BeWo cultures was unaffected by the presence of HDL3; findings which are in line with the low rate of selective CE-uptake from HDL3 in these cell lines (Fig 2) demonstrating that exogenous HDL3 may not significantly alter CE synthesis in these cell lines [45] Discussion Fig Identification of SR-BI on mRNA and protein level in human choriocarcinoma cells (A) RT-PCR analysis cDNA for SR-BI was amplified using specific forward and reverse oligonucleotide primers as described in Materials and methods The PCR product was separated on a 1.5% agarose gel: lane (BeWo), lane (JAr), lane (Jeg3), lane (bp standard) (B) Northern blot analysis Total RNA (15 lg) was subjected to agarose gel electrophoresis and hybridized using the 553 bp fragment as a probe: lane (BeWo), lane (JAr), lane (Jeg3), lane (total liver tissue) The blot was then stripped and reprobed by using a fragment from the human glyceraldehyde 3-phosohate dehydrogenase cDNA (Clontech) Conventional Northern blotting did not distinguish between SR-BI and SR-BII isoforms [27] (C) Western blot analysis Immunoblotting experiments of detergent solubilized membrane protein fractions (lane 1–3, 50 lg protein; lane 4, 10 lg protein) were separated on 8% SDS/PAGE Immunoreactive bands were visualized with rabbit anti-(SR-BI peptide) IgG (1 : 1500 dilution), peroxidase-conjugated goat anti-rabbit IgG, and the ECL-detection system Arrow indicates the 84-kDa position of SR-BI: lane (BeWo), lane (JAr), lane (Jeg3), lane [ldlA(mSR-BI)] Effects of exogenous HDL on growth rates To investigate whether (a) the pronounced cell association of [3H]CE-HDL3 and the high capacity for selective CEuptake from HDL3 can be shown during culture conditions and (b) to assess the role of exogenous cholesterol pool on cell proliferation of choriocarcinoma cells, the time-dependent effect of exogenous HDL3 on cellular cholesterol levels was measured (Fig 7A–C) While HDL3 had no significant effect on cell proliferation in BeWo cells, cell numbers were significantly (P > 0.05) increased in JAr and Jeg3 cells in the presence of HDL3 after 48 h Analysis of the cellular cholesterol content revealed that HDL3 led to a remarkable increase in cellular cholesterol levels in JAr and Jeg3 cells compared to cells cultured in the absence of HDL3 The During placental development the trophoblast cells develop along a cell lineage forming the villous cytotrophoblast with the overlaying syncytiotrophoblast, both responsible for hormone production and fetomaternal exchange (reviewed in ref [18]) In addition, another trophoblast population, the extravillous trophoblast of cell island and cell columns, is formed which maintains the ability of proliferation and invasion Choriocarcinoma is a malignant neoplasm that represents the early trophoblast of the attachment phase or as later invasive stage [46–48] Thus, in most cases, choriocarcinoma has the appearance of trophoblast, being predominantly syncytiotrophoblastic or cytotrophoblastic Some cytotrophoblastic choriocarcinomas secrete little human chorionic gonadotropin and some choriocarcinomas also secrete human placental lactogen [22–27] A number of choriocarcinoma cell lines have been established; these replicating trophoblasts, derived from the malignant tumor or produced by viral transformation of normal trophoblasts, are appropriate systems to mimick trophoblast behaviour in vitro Exogenous sources for cholesterol supply to fetal tissues involve receptor-mediated uptake of maternal LDL [2,17] Both trophoblasts [17,49] and trophoblast-like cells have been reported to bind LDL [17,20,21] Simpson et al [21] further reported that HDL3 was taken up and degraded by BeWo cells in a time- and concentration-dependent fashion, but the rate of degradation was considerably less than was the rate of degradation of LDL HDL serves as a physiological cholesterol/CE-carrier during reverse cholesterol transport from peripheral tissue to the liver and/or steroidogenic tissues As HDL may exert biological action in human trophoblast cells [50] we have investigated binding, cell association, and holoparticle association of HDL3 as well as selective CE-uptake from HDL3 in three different choriocarcinoma cells lines Binding of 125Ilabeled HDL3 (at °C) to BeWo, JAr, and Jeg3 cells studied here revealed Kd and bmax values (Table 1) comparable to those when binding of HDL3 to trophoblast membrane protein fractions [3,51] or to intact first trimester trophoblasts was investigated [52] However, the pronounced capability for lipid tracer uptake from CE-labeled HDL3 in excess of holoparticle association in JAr and Jeg3 (but not in BeWo) cells was indicative for SR-BI-mediated selective CE-uptake from HDL3 in these cell lines SR-BI has been identified in human placental tissues by Northern blot experiments [14] Initial attempts to explore a possible role of SR-BI on murine trophoblast cells have involved immunofluorescence microscopy to define the temporal and spatial pattern of SR-BI expression during rodent embryogenesis [16] Here, we provide evidence that SR-BI is highly expressed on human trophoblast-like choriocarcinoma cell lines JAr and Jeg3 and we further propose that HDL3 does act as cholesterol delivery vehicle to these cells via the SR-BI-mediated Ó FEBS 2003 458 C Wadsack et al (Eur J Biochem 270) Fig Immunocytochemical evidence for SR-BI on choriocarcinoma cells (A) BeWo, (B) Jar, (C) Jeg3, (D) ldlA(mSR-BI) and (E) ldlA7 cells were cultured on Laboratory-Tek chamber slides as described in Materials and methods Labeling was peformed with polyclonal rabbit anti-(SR-BI) IgG (dilution of : 1000) followed by goat anti-rabbit cyanine3 secondary antibody (dilution : 500) pathway The fact that the C-terminal cytosolic domain of SR-BI which is a critical domain involved in selective CE-uptake [29,53] is lacking in SR-BII and the lack of SR-BII protein in all three choriocarcinoma cell lines investigated here suggested that this receptor does not play a specific role for CE-supply during fetal development SR-BI may probably exert more profound capabilities than the common LDL-receptor pathway mediating holoparticle-uptake of apolipoprotein B- or E-containing lipoprotein particles This must be seen in light of the fact that expression of LDL-receptor mRNA decreases from first trimester to term tissues [54] and thus SR-BI could provide an alternative pathway for cholesterol/CE-supply during fetal development As SR-BI is critical for cholesterol transport its regulation has been addressed in a number of placental tissues involved in steroidogenesis [55–57] In humans, lacking functional LDL-receptor, fetal development and cholesterol supply is normal compared to controls Also in mice with a targeted deletion of the LDL-receptor gene, the up-regulation of selective CEuptake suggests that SR-BI can compensate for the loss of LDL-receptor function [58] Recently, the LDL-receptor related protein has also been addressed to mediate, at least in part, selective lipid uptake [59] LDL-receptor related protein was found to be associated with syncytiotrophoblasts Table Effect of HDL and ox–LDL on cell association of [3H]CEHDL at 37 °C Choriocarcinoma cells were incubated for h at 37 °C with 10 lgỈmL)1 of [3H]CE-labeled HDL3 in the absence (control) or presence of 100 lg of proteinỈmL)1 of unlabeled competitor (HDL or ox-LDL) in F12K (BeWo) or DMEM (JAr and Jeg3) The 100% value for cell association of [3H]CE-HDL was 105 ± ng (BeWo), 255 ± 17 (JAr) and 286 ± 21 ng HDL3-protein/mg cell protein (Jeg3) The cell association is expressed as the percentage of the radioactivity measured in the absence (100%) or presence of competitor Results are given as means ± SD of three independent experiments [3H]CE-HDL BeWo JAr Jeg3 Control HDL ox-LDL 100 100 100 43.5 ± 6.6 70.5 ± 9.5 79.4 ± 8.7 37.6 ± 5.7 21.2 ± 3.2 28.4 ± 4.3 Fig Effect of 8–CPTcAMP on cell association of [3H]CE-HDL and mRNA SR-BI expression in choriocarcinoma cells Following preincubation in F12K and DMEM without FBS (12 h) in the absence (open squares) or presence (full squares) of 0.3 mM 8-CPT-cAMP, choriocarcinoma cells were incubated for h with 10 lgỈmL)1 of [3H]CElabeled HDL3 at 37 °C The washed cells were lysed with 0.3 M NaOH to determine the cell-associated fraction To determine the in vitro effect of 8-CPT-cAMP on SR-BI mRNA expression, choriocarcinoma cells were incubated as described above Total RNA was isolated, and Northern blot analysis was performed using radiolabeled SR-BI cDNA probe for each cell line (top panel) in the absence (lane 1, 3, 5) or presence of 0.3 mM 8-CPT-cAMP (lane 2, 4, 6) Radiolabeled cDNA for GAPDH was used to monitor RNA loading (bottom panel) Results are given as means ± SD of three independent experiments (but not cytotrophoblasts) and BeWo cells [10] However, cAMP-treatment decreases expression of LDL-receptor related protein [10] but increases expression of SR-BI in parallel (Fig [13]) Ó FEBS 2003 Human choriocarcinoma cells and SR-BI (Eur J Biochem 270) 459 Fig Transfection of choriocarcinoma cells Untransfected (1, 4, and 7), mock-transfected (b-gal-transfected, 2, 5, and 8), and SR-BItransfected (3, 6, and 9) BeWo (1–3), JAr (4–6), and Jeg3 cells (7–9) were incubated for h with 10 lgỈmL)1 protein of [3H]CE-HDL3 at 37 °C as described in Fig Thereafter, the incubation medium was removed, the cells were washed and analyzed for cell-associated radioactivity of [3H]CE-HDL3 Results are given as means ± SD of three independent experiments Immunoblot experiments of detergent solubilized membrane protein fractions (50 lg protein per lane) of untransfected (1, 4, and 7) and SR-BI-transfected cells (3, 6, and 9) were performed as described in Fig 3C Different lines of evidence are of support that SR-BI is a physiologically relevant HDL receptor, studies which are primarily addressing its role in cholesterol metabolism [12,13] Further studies demonstrated that SR-BI-deficiency in rodents leads to defective erythroid maturation and abnormalities in the female reproductive system [13,60] Evidence is accumulating that cholesterol must be considered as an essential agent in embryonic development Histochemical analysis of ovaries from superovulated females showed reduced oil red O staining of lipids in the ovarian copora lutea of SR-BI knock out relative to those of wild-type animals suggesting reduced CE storage as a source for steroid hormone production [61] Also plasma progesterone levels between pseudopregnant controls and knock out females days after mating were slightly although not significantly impaired Finally, the majority of embryos from SR-BI knock out females at harvesting showed abnormal, nonrefractile morphology of oocytes and embryos [61] similar to wild-type females that had been treated with cholesterol-binding drugs that can perturb membrane structure Female mice with a targeted null mutation of the SR-BI gene are infertile [61], a fact underscoring the importance of the SR-BI pathway during embryonic development As SR-BI is expressed on the maternal–fetal interface [16], this could indicate defective cholesterol/CE-supply to the growing embryo From our studies we propose that some choriocarcinoma cell lines can be used as a suitable model to mimick Fig Effect of exogenous HDL on cellular growth rates and cholesterol content Cells were seeded in 6-well trays and incubated in F12K (BeWo) and DMEM (JAr and Jeg3) containing 10% lipoproteindeficient serum (open symbols) or in medium containing lipoproteindeficient serum and 100 lgỈmL)1 HDL3 (closed symbols) At the indicated time points the cells were washed with HBBS, trypsin-treated and the cell number was counted The cellular cholesterol content of lysed cells was analysed with enzymatic cholesterol reagent Data shown represent means ± SD of three independent experiments (A) BeWo, (B) JAr, (C) Jeg3 Ó FEBS 2003 460 C Wadsack et al (Eur J Biochem 270) trophoblast–lipoprotein interactions in vitro In BeWo the LDL-receptor seems to be the predominant pathway supplying these cells with cholesterol/CE via holoparticleuptake [10] The fact that BeWo, a cell line with many characteristics of cytotrophoblast, are almost lacking SR-BI and SR-BI-mediated selective CE-uptake from HDL3, supports the assumption that JAr and Jeg3 resembling syncytiotrophoblasts-like properties are suitable in vitro models to study directed cholesterol/CE-transport in polarized cells Invasiveness of trophoblast cells in vivo and choriocarcinoma cells in vitro apparently is linked to celldifferentiation and proliferation [47] similar to that reported for other tumors [62,63] In line with previous findings using various differentiation-modulating agents [47,48] we also observed pronounced differences between BeWo and JAr/Jeg3 cells regarding proliferation rates The fact that exogenous HDL3 remarkably increases proliferation in JAr and Jeg3 supports a receptor-mediated specific and selective CEuptake and cellular growth in parallel The results of this study demonstrate that JAr and Jeg3 cells, replicating trophoblast cells derived from a malignant tumor are appropriate cellular systems to mimick cholesterol supply from maternal lipoproteins to developing embryonic tissues via SR-BI-mediated CE-uptake from HDL3 10 11 12 13 14 15 Acknowledgements The authors thank Dr Vardon (local blood bank) providing human plasma This work was supported by the Austrian Science Fund (FWF, 15404 to E M and SFB 007–716 to W S.) and the Austrian National Bank OENB 8840 (to A H.), and 8778 and 9962 (to E M.) 16 17 References Woollett, L.A (2001) The origins and roles of cholesterol and fatty acids in the fetus Curr Opin Lipidol 12, 305–312 Alsat, E., Bouali, Y., Goldstein, S., Malassine, A., 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scavenger receptor class B type I Endocrinology 142, 4540–4549  ... the early trophoblast of the attachment phase or as later invasive stage [46–48] Thus, in most cases, choriocarcinoma has the appearance of trophoblast, being predominantly syncytiotrophoblastic... cytotrophoblastic Some cytotrophoblastic choriocarcinomas secrete little human chorionic gonadotropin and some choriocarcinomas also secrete human placental lactogen [22–27] A number of choriocarcinoma. .. was aspirated and the cells were rinsed as described above [34] Degradation of 125I-labeled HDL3 by choriocarcinoma cells was estimated by measuring the nontrichloroacetic acid-precipitable radioactivity