MRP2, a human conjugate export pump, is present and transports fluo 3 into apical vacuoles of Hep G2 cells

Tobias Cantz1, Anne T. Nies1, Manuela Brom1, Alan F. Hofmann2, and Dietrich Keppler1

1 Division of Tumor Biochemistry, Deutsches Krebsforschungszentrum, D-69120 Heidelberg, Germany; and 2 Division of Gastroenterology, Department of Medicine, University of California, San Diego, California 92093-0813


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The multidrug resistance protein 2 (MRP2, symbol ABCC2) transports anionic conjugates and certain amphiphilic anions across the apical membrane of polarized cells. Human hepatoma Hep G2 cells retain hepatic polarity and form apical vacuoles into which cholephilic substances are secreted. Immunofluorescence microscopy showed that human MRP2 was expressed in the apical vacuole membrane of polarized Hep G2 cells, whereas the isoform MRP3 was localized to the lateral membrane. Expression of both MRP2 and MRP3 was confirmed by immunoblotting and reverse transcription PCR. Fluo 3 secretion into the apical vacuoles was inhibited by cyclosporin A but not by selective inhibitors of multidrug resistance 1 P-glycoprotein. In addition, carboxyfluorescein, rhodamine 123, and the fluorescent bile salt derivatives ursodeoxycholyl-(Nepsilon -nitrobenzoxadiazolyl)-lysine and cholylglycylamido-fluorescein were secreted into the apical vacuoles; the latter two probably via the bile salt export pump. We conclude that MRP2 mediates fluo 3 secretion into the apical vacuoles of polarized Hep G2 cells. Thus the function of human MRP2 and the action of inhibitors can be analyzed by the secretion of fluorescent anions such as fluo 3.

multidrug resistance protein 2 (ABCC2); multidrug resistance protein 3 (ABCC3); ATP-dependent transport; fluorescent bile salts; cyclosporin A


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE CANALICULAR MEMBRANE OF the hepatocyte contains different transport proteins that mediate the ATP-dependent secretion of organic cations [multidrug resistance (MDR) 1 P-glycoprotein], phospholipids (MDR3 P-glycoprotein), bile salts [bile salt export pump (BSEP), also known as sister of P-glycoprotein], and certain conjugates and amphiphilic anions [multidrug resistance protein (MRP) 2] into bile (for review, see Ref. 28). The apical conjugate export pump MRP2 was recently cloned from human and rat tissues and initially termed canalicular multidrug resistance protein (cMRP) (6) or canalicular multispecific organic anion transporter (cMOAT) (20, 41, 50). Because of similar substrate specificities (9, 29) and related amino acid sequences (6, 20, 41, 50) of MRP2 and MRP1, the term MRP2 is used here for this 190-kDa membrane glycoprotein. The first cloned member of the multidrug resistance protein family was MRP1, which was cloned from a small lung cancer cell line (8). MRP2 was detected in the apical membrane of polarized cells, such as rat and human hepatocytes (6, 34, 42), renal proximal tubule cells (46, 47), and rat hepatoma-derived WIF-B cells (36). Recently, another MRP isoform, the MRP3, was cloned from liver and localized to the basolateral membrane of human hepatocytes (34).

Two mutant rat strains, the Groningen yellow/transport-deficient (GY/TR-) and the Eisai hyperbilirubinemic (EHBR) rats, have been described that have a hereditary defect in the hepatobiliary excretion of anionic conjugates (18, 21, 39, 49). The use of inside-out oriented canalicular membrane vesicles isolated from normal rat hepatocytes in comparison with those from transport-deficient hepatocytes from mutant GY/TR- or EHBR rats suggested an ATP-dependent transport system for amphiphilic anions across the bile canalicular membrane (6, 19, 37). Cloning of the MRP2 protein from normal rat liver revealed that lack of transport activity in the mutant rat strains is due to absence of the MRP2 protein (6, 20, 41). In humans, the lack of the MRP2 protein in the canalicular membrane is the molecular basis of the Dubin-Johnson syndrome (27, 42) and leads to conjugated hyperbilirubinemia.

Fluorescent compounds are frequently used to study transporter activity in intact cells, e.g., rhodamine 123 to detect MDR1 P-glycoprotein transport activity (51, 55). Bile salts tagged with a fluorophore have been used to follow bile salt secretion into bile in the biliary fistula rat (16) or the isolated perfused rat liver (15). To investigate MRP2 transport activity in intact cells, chloromethylfluorescein diacetate (CMFDA; Refs. 40 and 44) and monochlorobimane (38, 43) have been used. CMFDA and monochlorobimane are intracellularly converted to their respective fluorescent glutathione conjugates: glutathionylmethylfluorescein (2) and glutathionylbimane (2). Glutathionylbimane accumulates in intracellular vesicles of intact hepatocytes isolated from normal liver but not in those from MRP2-deficient mutant rat livers lacking the MRP2 transporter (38, 43). Similarly, CMFDA is secreted into bile from normal rats but not into that from mutant GY/TR- rats (40, 44). Recently, two other fluorescent organic anions were used for studying MRP-mediated transport. 5-Carboxyfluorescein diacetate (5-CFDA) is a nonfluorescent substrate that is intracellularly converted to its fluorescent form 5-carboxyfluorescein (CF; Ref. 5) but not intracellularly conjugated to glutathione (2). CF was suggested as a substrate for MRP1 and presumably also for MRP2 (54). Furthermore, we have shown previously that the fluorescent amphiphilic anion fluo 3 is a substrate of relatively high affinity for rat MRP2 and that rat MRP2 mediates secretion of fluo 3 into apical vacuoles of rat hepatoma WIF-B cells (36). The penta-AM of fluo 3, fluo 3-AM, is membrane permeable and therefore used to load cells (26). Free anionic fluo 3 is then formed by intracellular esterases. Because the fluorescence of fluo 3 is largely increased on Ca2+ binding, fluo 3 has been widely used as an intracellular Ca2+ indicator (26, 35).

In this study, we investigated localization and function of human MRP2 in the hepatoblastoma Hep G2 cell line. Hep G2 cells retain hepatic polarity and form bile canaliculus-like structures (48), termed apical vacuoles, into which fluorescent amphiphilic anions such as glutathionylmethylfluorescein are secreted (45). Immunofluorescence analysis and secretion of fluo 3 and CF into the apical vacuoles indicated that functional human MRP2 is localized in the apical membrane of polarized Hep G2 cells. Fluo 3 secretion was characterized by different inhibitors of MRP2- and P-glycoprotein-mediated secretion. The apical membrane of Hep G2 cells was also found to contain transporters for secretion of the fluorescent compound rhodamine 123 and the fluorescent bile salt derivatives ursodeoxycholyl-(Nepsilon -nitrobenzoxadiazolyl)-lysine (UDC-L-NBD) and cholylglycylamidofluorescein (CGamF).


    MATERIALS AND METHODS
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MATERIALS AND METHODS
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Chemicals. Fluo 3-AM, rhodamine 123, and 5-CFDA were obtained from Molecular Probes (Eugene, OR). The fluorescent bile salt derivatives UDC-L-NBD and CGamF were prepared as described previously (16). Cyclosporin A in its preparation for clinical use (Sandimmun) and its nonimmunosuppressive analog 3'-oxo-4-butenyl-4-methyl-(Thr1)-(Val2)-cyclosporin (PSC833; Ref. 3) were from Sandoz (Basel, Switzerland). The selective MDR1 P-glycoprotein inhibitor (2r)-anti-5-[3-[4-(10,11-difluoromethanodibenzo-suber-5-yl)- piperazin-1-yl]-2-hydroxy-propoxy]quinoline trihydrochloride (LY-335979; Ref. 10) was a gift from the Eli Lilly Research Laboratories (Indianapolis, IN). Cremophor EL was obtained from Sigma (Deisenhofen, Germany). Microgrid glass coverslips (Cellocate) were from Eppendorf (Hamburg, Germany). All other chemicals were of analytical grade and obtained either from Merck (Darmstadt, Germany) or Sigma.

Antibodies. The polyclonal rabbit antibody EAG5 was raised against the carboxy terminus of human MRP2 (6) and used as described previously (23). The polyclonal rabbit antibody FDS was raised against the carboxy terminus of human MRP3 (34). Mouse monoclonal antibody C219 directed against P-glycoprotein was from Centocor (Antwerp, Belgium), and mouse monoclonal CD26 antibody against the human ectoenzyme dipeptidylpeptidase IV (anti-DPPIV) was from Dianova (Hamburg, Germany). The mouse monoclonal MRP1-specific antibody QCRL-1 (14) was kindly provided by Dr. R. G. Deeley and Dr. S. P. C. Cole (Queen's University, Kingston, ON, Canada). Goat anti-rabbit secondary antibody coupled to tetramethylrhodamine isothiocyanate (TRITC) was from Sigma, and goat anti-mouse secondary antibody coupled to Cy2 was from Dianova.

Cell culture. The human hepatoma cell line Hep G2 was provided by the Tumorbank of Deutsches Krebsforschungszentrum (Heidelberg, Germany) and maintained as described previously (23). Cells were seeded onto glass coverslips at a density of ~5 × 104 cells/cm2. Cells were usually used after 5-7 days of culture, when confluency and maximal polarity had been reached. At this time, intercellular phase-lucent structures were visible that have been described as bile canaliculus-like spaces (48) or apical vacuoles (45). MRP1-transfected HeLa T5 cells were kindly provided by Dr. R. G. Deeley and Dr. S. P. C. Cole and cultured as described previously (23). Madin-Darby canine kidney cells (MDCK cells) expressing MRP3 were also cultured as described previously (34).

RNA isolation and reverse transcription PCR. RNA was isolated from Hep G2 cells grown to confluency using the High Pure RNA isolation kit (Roche Molecular Biochemicals, Mannheim, Germany). Reverse transcription and PCR were carried out as described previously (36). The following primers were used: for human MRP1 (GenBank/EMBL accession no. L05628), sense primer 5'-AGGCCTATTACCCCAGCAT-3' (bases 3560 to 3578) and antisense primer 5'-CGATCTTGGCGATGTTGATG-3' (bases 4084-4065); for human MRP2 (GenBank/EMBL accession no. X96395), sense primer 5'-AATAGCACCGACTATCCA-3' (bases 3031 to 3048) and antisense primer 5'-GTGGGATAACCCAAGTTG-3' (bases 4284 to 4267); for human MRP3 (GenBank/EMBL accession no. Y17151), sense primer 5'-TGAGATCATCAGTGATACTAA-3' (bases 3510 to 3530) and antisense primer 5'-ATGCGGCTCTTGCGGAG-3' (bases 4337 to 4321). The beta -actin control primer pair was from Stratagene (Amsterdam, The Netherlands). PCR cycling conditions were as follows: 94°C for 60 s and primer annealing for 60s, elongation at 72°C, for a total of 35 cycles (MRP1, MRP2, MRP3) and 28 cycles (beta -actin), respectively. Amplification was terminated by a final incubation at 72°C for 10 min. Annealing temperatures were 49°C for MRP2, 58°C for MRP1 and MRP3, and 60°C for beta -actin. The PCR products were separated on 1% agarose gels and visualized by ethidium bromide staining. Subcloning and sequencing of PCR fragments were done as described previously (36).

Membrane preparation and immunoblotting. Plasma membrane vesicles were prepared from HeLa T5 cells as described previously (23). Crude membrane fractions were prepared from Hep G2 and MRP3-expressing MDCK cells as described previously (34). Proteins were separated on 7.5% SDS-polyacrylamide gels and immunoblotted on nitrocellulose membranes using a tank blotting system (BioRad, Munich, Germany). Bands were visualized by enhanced chemiluminescence detection (Amersham-Pharmacia, Freiburg, Germany) as described previously (36).

Indirect immunofluorescence microscopy. Immunofluorescence staining of Hep G2 cells grown on glass coverslips was carried out as described previously for hepatoma-derived WIF-B cells (36) using the following primary antibody dilutions (in PBS): 1:150 for EAG5, 1:100 for FDS, 1:15 for QCRL-1, 1:1,500 for anti-DPPIV, and 1:100 for C219. TRITC-labeled goat anti-rabbit IgG (1:200 in PBS) and Cy2-labeled goat anti-mouse IgG (1:400 in PBS) were used as the secondary antibodies. Fluorescent staining of cells was observed on an Axiovert S100TV microscope (Carl Zeiss, Jena, Germany) equipped with appropriate filter combinations using a 40× oil immersion lens. Pictures were taken with a digital camera (Hamamatsu, Hamamatsu, Japan), analyzed by Openlab Imaging Software (Improvision, Coventry, UK), and processed and printed using Picture Publisher and Designer (Micrografx, Richardson, TX).

Secretion of fluorescent substrates into apical vacuoles of Hep G2 cells. Fluo 3-AM was dissolved in DMSO as described previously (36). 5-CFDA was prepared as a 2-mM stock solution in ethanol. CGamF and rhodamine 123 were dissolved in DMSO to give 10-mM stock solutions. The exact concentration of rhodamine was determined by measuring the absorption at 507 nm using a molar extinction coefficient of 101 cm2/µmol, and that of CGamF was determined by measuring the absorption at 492 nm using a molar extinction coefficient of 80 cm2/µmol (15). UDC-L-NBD was dissolved in ethanol to give a 10-mM stock solution.

Polarized Hep G2 cells grown on microgrid glass coverslips were loaded in medium with 1 µM fluo 3-AM, 2 µM 5-CFDA, 10 µM UDC-L-NBD, 20 µM CGamF, or 2.5 µM rhodamine 123 for 15 min at 37°C. This time period proved to be optimal for intracellular formation of the fluorescent substances and their secretion into the apical vacuoles. In some experiments, cells were preincubated with 1 µM LY-335979 for 1 h at 37°C and washed with medium before loading with rhodamine 123. After loading, coverslips were washed in medium and placed into a cell chamber (Attofluor; Molecular Probes) filled with medium to ensure viability of the cells. Pictures of apical vacuoles filled with fluorescent substrate were taken on the Axiovert S100TV microscope as described in Indirect immunofluorescence microscopy. After careful removal of the coverslips from the cell chamber, cells were fixed and indirect immunofluorescence staining was carried out using the EAG5 antibody for identification of the substrate-filled structures as apical vacuoles of Hep G2 cells. The secondary antibody was TRITC-labeled goat anti-rabbit IgG (1:200 in PBS). Immunofluorescence micrographs matching the corresponding micrographs of the fluorescent substrate-filled apical vacuoles were taken. In some experiments, Hep G2 cells were simultaneously incubated for 30 min at 37°C with either fluorescent bile salt or LysoTracker Red (Molecular Probes) to visualize intracellular vesicles with acidic pH.

Inhibition of secretion of fluorescent substrates into apical vacuoles of Hep G2 cells. The cyclosporin A preparation (50 mg/ml) contained 33% ethanol, castor oil, and polyoxyethylene (650 mg/ml). PSC833 and LY-335979 were prepared in ethanol as 10 mM and 3.2 mM stock solutions, respectively. In control experiments without cyclosporin A, PSC833, or LY-335979, cells were incubated with Cremophor EL (solvent for cyclosporin A) or ethanol (solvent for PSC833 and LY-335979) to exclude effects of the solvent.

For analysis of fluo 3 secretion, Hep G2 cells were grown on microgrid coverslips until reaching confluency. Cells were incubated in medium with different concentrations of the inhibitors for 2 h at 37°C (0, 5, and 10 µM for cyclosporin A and LY-335979 and 0, 5, 10, and 50 µM for PSC833). To remove extracellular inhibitors that might interfere with the uptake of fluo 3-AM, the cells were washed in medium and subsequently loaded with 1 µM fluo 3-AM for 15 min at 37°C. Cells were then washed in cold PBS and observed on a fluorescence microscope (Zeiss Axioskop). Fluorescent, fluo 3-filled apical vacuoles were counted in 25 fields, each with a square size of 350 × 350 µm. The number of fluorescent apical vacuoles in the presence of inhibitor (duplicates) is expressed as percentage of the number of fluorescent apical vacuoles in the absence of inhibitor (quadruplicates).

For analysis of the secretion of further fluorescent substrates, Hep G2 cells were seeded onto normal glass coverslips. Confluent cultures were incubated in media with different inhibitor concentrations at 37°C for 2 h. The cells were then washed in medium and simultaneously loaded with the substrate (2 µM 5-CFDA, 10 µM UDC-L-NBD, and 20 µM CGamF) and 1 µg/ml of the fluorescent nuclear stain Hoechst 33258 (Molecular Probes) for 15 min at 37°C. Cells were washed three times with cold PBS and observed on a fluorescence microscope. First, the number of cells, as visualized by the nuclear Hoechst 33258 stain, and subsequently the number of fluorescent, substrate-filled apical vacuoles were counted. For each substrate and inhibitor concentration, the ratio of the number of apical vacuoles to the number of total cells was calculated. The ratio in the presence of inhibitor is expressed as percentage of the ratio in the absence of inhibitor. For each concentration, three independent cultures and at least 500 cells were analyzed.


    RESULTS
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Immunofluorescence localization of MRP2 and MRP3 in Hep G2 cells. The EAG5 antibody resulted in intense fluorescence of ring-like structures along the circumference of apical vacuoles (Fig. 1A), which can also be detected by phase contrast (Fig. 1B), demonstrating localization of MRP2 in the apical membrane of Hep G2 cells. Double-label experiments with EAG5 and the anti-DPPIV antibody or EAG5 and the C219 antibody, which recognizes P-glycoprotein, were performed to further define the localization of the structures recognized by the EAG5 antibody in Hep G2 cells. DPPIV (17) and P-glycoprotein (52) are both found as well in the canalicular membrane of hepatocytes. Superimposition of the red EAG5-stained structures (Fig. 1C) with the green ring-like DPPIV fluorescence (Fig. 1D) or the green ring-like P-glycoprotein fluorescence (not shown) resulted in a yellow color (Fig. 1E), indicating colocalization of MRP2 with DPPIV and P-glycoprotein in the apical membrane of Hep G2 cells. However, double-label experiments with FDS (Fig. 1F), recognizing human MRP3, and the anti-DPPIV antibody (Fig. 1G) and the subsequent merging of both fluorescences (Fig. 1H) indicated that MRP3 and DPPIV localize to different plasma membrane domains in Hep G2 cells. With the use of the monoclonal QCRL-1 antibody, which is directed against MRP1 (14), weak lateral plasma membrane staining was observed in ~10% of the Hep G2 cells, indicating low expression of MRP1 in Hep G2 cells. In contrast, strong plasma membrane staining of MRP1-expressing HeLa T5 cells was observed.


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Fig. 1.   Double-label immunofluorescence analysis of multidrug resistance protein (MRP) 2 and MRP3 localization in polarized Hep G2 cells. Micrographs of confluent cultures grown on glass coverslips are shown after reaction with polyclonal antibody EAG5 (red; A and C), raised against carboxy terminus of human MRP2 (6, 23), and FDS (red; F), raised against carboxy terminus of human MRP3 (34), compared with reaction of an antibody detecting dipeptidylpeptidase IV (DPPIV) (green; D and G). Superimposition of corresponding micrographs demonstrates colocalization of MRP2 and DPPIV (E) in apical membrane, which forms phase-lucent apical vacuoles between two or more Hep G2 cells (as seen in phase-contrast microscopy; B) and localization of MRP3 and DPPIV to different plasma membrane domains (H). Arrowheads point to apical vacuoles. Bars = 10 µm.

Immunoblot detection of MRP isoforms. Expression of MRP2 and MRP3 in Hep G2 cells was confirmed by immunoblot analysis (Fig. 2) using the antibodies EAG5, directed against MRP2 (6), and FDS, directed against MRP3 (34). In addition, the monoclonal antibody QCRL-1 against MRP1 (14) was used. The EAG5 antibody detected MRP2 in Hep G2 cells as a 190-kDa protein. With the FDS antibody, two immunoreactive bands at 190 kDa and 170 kDa were detected. The same signal pattern was observed in human liver, in which MRP3 is present in two differentially spliced variants (34). With the use of the QCRL-1 antibody, no immunoreactive bands were detected in Hep G2 cell membranes. This finding was consistent with the previously reported very low expression of MRP1 in the Hep G2 cells under our culture conditions (23). With the use of immunoblot analysis, the QCRL-1 antibody did not detect any bands in homogenates from MRP3-expressing MDCK cells.


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Fig. 2.   Immunodetection of MRP isoforms in Hep G2 cells. Plasma membrane vesicles from MRP1-expressing HeLa T5 cells (5 µg) and membrane fractions from Hep G2 cells (50 µg) were separated on SDS polyacrylamide gels and transferred to nitrocellulose membranes. MRP2 expression was detected using EAG5 antibody (6, 23), and MRP3 expression was detected using FDS antibody (34). MRP1 was not detected in Hep G2 membrane fractions, even after long exposure of nitrocellulose membrane.

Detection of mRNA of MRP isoforms. Expression of the human MRP2 and the human MRP3 gene was also confirmed by PCR amplification of cDNA generated from reverse-transcribed mRNA (Fig. 3). Primers corresponding to the human MRP2 sequence (6, 30) yielded a fragment of the expected size of 1,254 bp. This MRP2 cDNA fragment was identified by subcloning and sequencing of 200 nucleotides. The sequence was identical to the corresponding sequence of human liver MRP2 (6, 30). Similarly, PCR amplification with primers corresponding to the human MRP3 sequence (34) yielded a fragment of the expected size of 828 bp. Sequencing of this fragment confirmed identity to the corresponding sequence of human liver MRP3 (34). In addition to MRP2 and MRP3 mRNA, MRP1 mRNA was also detectable when using primers corresponding to the human MRP1 sequence (8).


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Fig. 3.   Detection of MRP1, MRP2, and MRP3 mRNA in Hep G2 cells by reverse transcription PCR analysis. Reverse transcription was performed on Hep G2 total RNA with an oligo(dT)18 primer yielding single-stranded cDNAs. For PCR analysis, 3 primer pairs specific for human MRP1 mRNA (525-bp fragment), human MRP2 mRNA (1,254-bp fragment) and human MRP3 mRNA (828-bp fragment) were used. Control experiments assaying amplification of genomic DNA were negative.

Secretion of fluo 3 and additional fluorescent substrates into apical vacuoles of Hep G2 cells. The function of MRP2 in the apical membrane of Hep G2 cells was studied by following the secretion of the fluorescent organic anions fluo 3 and CF. After loading the cells with the membrane-permeable, nonfluorescent substances fluo 3-AM or 5-CFDA, fluorescent apical vacuoles were visible, indicating that either substance had been taken up into the cells and cleaved to its fluorescent form, which was subsequently secreted into the apical vacuoles (Fig. 4, A, C, and F). To verify that the substrate-filled structures were indeed apical vacuoles of Hep G2 cells, cells were stained with the EAG5 antibody. Merging of the green fluo 3 fluorescence (Fig. 4C) with the red EAG5 immunofluorescence (Fig. 4D) confirmed that the fluo 3-filled structure was an apical vacuole (Fig. 4E). Similar results were obtained after merging green CF fluorescence and red EAG5 immunofluorescence (Fig. 4, F-H).


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Fig. 4.   Vectorial transport of fluo 3 and carboxyfluorescein (CF) into apical vacuoles of Hep G2 cells. Polarized, confluent cultures were incubated for 15 min at 37°C in complete medium with 1 µM fluo 3-AM (A-E), or 2 µM 5-CF diacetate (5-CFDA; F-H). After incubation, cells were washed with medium and substrate-filled vacuoles were observed by epifluorescence microscopy (green; A, C, and F). Subsequently, cells were fixed and immunostained with polyclonal antibody EAG5 (red; D and G) to visualize MRP2-positive structures, i.e., apical vacuoles. Merging red EAG5 fluorescence with green fluorescence of substrate-filled vacuoles demonstrates secretion of fluorescent substrates into apical vacuoles of Hep G2 cells (E and H). Bars = 10 µm.

Three other fluorescent substances were analyzed for their property of being secreted into apical vacuoles of Hep G2 cells: CGamF, a fluorescent derivative of the bile salt cholylglycine (16), UDC-L-NBD, a fluorescent derivative of the bile salt ursodeoxycholate (16), and rhodamine 123, which has also been identified as a substrate for MDR1 P-glycoprotein (51, 55). Apical vacuoles of Hep G2 cells were again visualized by EAG5 immunostaining (Fig. 5). After 15 min of incubation, the two fluorescent bile salts UDC-L-NBD (Fig. 5, A and C) and CGamF (Fig. 5, B and D) were secreted into the apical vacuoles of Hep G2 cells. In addition, UDC-L-NBD was also sequestered into intracellular punctate structures, whereas CGamF was observed mainly in the apical vacuoles (Fig. 5, A and B). Some of the structures in which UDC-L-NBD accumulated were acidic, as observed by simultaneous accumulation of the acidophilic LysoTracker Red. Rhodamine 123 was also accumulated in mitochondria (see also Ref. 24) in addition to apical vacuoles (Fig. 5E). LY-335979, a selective and potent MDR1 P-glycoprotein inhibitor (10), inhibited rhodamine 123 secretion into apical vacuoles (Fig. 5F). At concentrations >1 µM, no rhodamine 123-filled apical vacuoles were detected, whereas the mitochondrial staining by rhodamine 123 remained unchanged.


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Fig. 5.   Vectorial transport of fluorescent bile salt derivatives and rhodamine 123 into apical vacuoles of Hep G2 cells. Polarized, confluent cultures were incubated for 15 min at 37°C in complete medium with 10 µM ursodeoxycholyl-(Nepsilon -nitrobenzoxadiazolyl)-lysine (UDC-L-NBD; A and C), 20 µM cholylglycylamido-fluorescein (CGamF; B and D), 2.5 µM rhodamine 123 (E), or with 1 µM LY-335979 and subsequently with 2.5 µM rhodamine 123 (F). After incubation, substrate-filled vacuoles were observed by epifluorescence microscopy (green; A-F). Cells were then fixed and immunostained with EAG5 antibody (red) to visualize MRP2-positive structures, i.e., apical vacuoles (C-F). Preincubation of cells with selective MDR1 P-glycoprotein inhibitor LY-335979 (10) abolished secretion of the P-glycoprotein substrate rhodamine 123 into apical vacuoles (F); however, mitochondrial staining was still observed. Red fluorescence in A and B shows accumulation of LysoTracker Red, indicating presence of intracellular structures with an acidic pH. Yellow indicates presence of UDC-L-NBD in acidic organelles (arrows). Arrowheads point to apical vacuoles. Bars = 10 µm.

Inhibition of secretion of fluorescent substances into apical vacuoles of Hep G2 cells. Because not all structures staining positive for MRP2 in immunofluorescence microscopy were visible by phase contrast, as is, for example, the case in rat hepatoma-derived WIF-B cells, the previously described approach (36) of comparing the number of substrate-filled vacuoles with the number of phase-lucent structures was not applicable. As described in METHODS, we therefore used two methods to count fluorescent vacuoles under standardized conditions and to analyze the effect of different substances on secretion of fluorescent substrates into apical vacuoles of Hep G2 cells.

Increasing concentrations of cyclosporin A in the medium resulted in a decrease in the number of fluo 3- containing fluorescent apical vacuoles. In the presence of 10 µM cyclosporin A, the number of fluorescent vacuoles was reduced to 50%, compared with the number in the absence of cyclosporin A (Fig. 6). The nonimmunosuppressive cyclosporin derivative PSC833 had a weaker inhibitory effect than cyclosporin A. At 10 µM PSC833 in the medium, 76 ± 8% of apical vacuoles were filled with fluo 3. A concentration of ~50 µM was needed to cause a 56 ± 4% (n = 3) reduction in the number of fluorescent fluo 3-filled vacuoles. The selective MDR1 P-glycoprotein inhibitor LY-335979 (10 µM) had no significant effect on fluo 3 secretion. Cyclosporin A also inhibited CF secretion; at 10 µM, the number of fluorescent vacuoles was reduced to ~50% compared with the number in the absence of cyclosporin A (Fig. 6).


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Fig. 6.   Quantitative analysis of the inhibition of fluo 3 (A-C), CF (D), CGamF (E), and UDC-L-NBD (F) secretion into apical vacuoles of Hep G2 cells by cyclosporin A (A and D-F), its nonimmunosuppressive analog PSC833 (B), and selective P-glycoprotein inhibitor LY-335979 (C). For analysis of fluo 3 secretion, cells were grown on microgrid coverslips until reaching confluency and incubated with cyclosporin A, PSC833, or LY-335979 at indicated concentrations for 2 h at 37°C. Subsequent incubation with fluo 3-AM was performed as described in Fig. 4 legend. After rinsing in cold PBS, cells were observed by epifluorescence microscopy and number of fluorescent apical vacuoles in 25 fields (350 × 350 µm) was determined. Data are means ± SD of 3 independently analyzed cultures in absence of inhibitors (quadruplicates, 100%) or in presence of inhibitors (duplicates). Cremophor EL (vehicle of cyclosporin A; 100 ± 9%) and ethanol (94 ± 3%) had no significant effect on fluo 3 secretion. For analysis of secretion of CF, CGamF, and UDC-L-NBD, cells were grown on coverslips and incubated with different concentrations of cyclosporin A and then simultaneously with fluorescent substrate and nuclear Hoechst 33258 stain. Number of cells and number of fluorescent, substrate-filled apical vacuoles were counted and ratio of number of apical vacuoles to number of total cells was calculated. Ratio in presence of inhibitor is expressed as percentage of ratio in absence of inhibitor. For each concentration, 3 independent cultures and at least 500 cells were analyzed.

The effect of cyclosporin A on CGamF and UDC-L-NBD secretion was also analyzed. Increasing concentrations of cyclosporin A in the medium led to a decrease in the number of bile salt-filled apical vacuoles. In the case of CGamF, the presence of 0.5 µM and 2 µM cyclosporin A reduced the number of fluorescent apical vacuoles to 64 ± 8% and 37 ± 5%, respectively, of control values (Fig. 6). A 50% reduction of UDC-L-NBD-filled vacuoles was observed in the presence of 2 µM cyclosporin A.

Incubation of cells with the inhibitors might cause a decrease in cell viability and in density of the apical vacuoles. We therefore determined the ratios of the number of apical vacuoles to the number of cells in the absence and presence of different inhibitors. The ratios were 0.27 ± 0.02 (control, no inhibitor), 0.25 ± 0.04 (10 µM cyclosporin A), 0.27 ± 0.01 (10 µM PSC833), and 0.26 ± 0.02 (10 µM LY-335979), respectively, indicating that neither inhibitor had a significant effect on the density of apical vacuoles.


    DISCUSSION
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ABSTRACT
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DISCUSSION
REFERENCES

The human apical conjugate export pump MRP2 belongs to the MRP family and mediates the ATP-dependent transport of anionic conjugates across membranes (6, 9, 29). MRP2 is predominantly localized to the apical membrane of polarized cells, such as hepatocytes (6, 34), rat hepatoma-derived cells (36), and proximal tubule epithelia of the kidney (46, 47). To study the function of human MRP2 in intact cells, we used the human hepatoma Hep G2 cell line with retained hepatic polarity (48). Immunofluorescence microscopy (Fig. 1), immunoblot analysis (Fig. 2), reverse transcription PCR analysis (Fig. 3), and transport studies in intact cells (Figs. 4-6) demonstrated apical localization and functional expression of human MRP2 in this system. Our results indicate that the Hep G2 cell line is most useful for studies on human MRP2 and its transport function.

Expression of MRP2 and MRP3 in Hep G2 cells. Hep G2 cells were derived from a human hepatoma (1); however, despite their malignant transformation these cells have retained many hepatocyte-specific functions such as albumin and bile acid synthesis (22, 31). Furthermore, after several days in culture, Hep G2 cells develop hepatic polarity with bile canaliculus-like structures (48), visible as phase-lucent structures between adjacent cells (Fig. 1). The membrane surrounding these apical vacuoles is structurally and functionally analogous to the canalicular membrane in forming microvilli (48) and expressing at least some canalicular proteins, e.g., the P-glycoprotein (45).

Expression of the conjugate export pump MRP2 in Hep G2 cells has been indicated previously by immunoblotting and ATP-dependent transport of leukotriene C4 and bilirubin glucuronides into inside-out oriented membrane vesicles (23). Reverse transcription PCR analysis using MRP2-specific primers confirmed the expression of MRP2 in Hep G2 cells (Fig. 3). Double-label immunofluorescence studies with antibodies recognizing the apical plasma membrane proteins DPPIV (17) or P-glycoprotein (52) and an antibody selectively recognizing MRP2 revealed that MRP2 is expressed in the apical membrane of Hep G2 cells (Fig. 1). This finding is consistent with the apical localization of MRP2 in hepatocytes (6, 34) and proximal tubule epithelial cells (46, 47). Localization of MRP2 in the apical membrane of Hep G2 cells supports the view that this membrane is functionally and compositionally analogous to the canalicular membrane of hepatocytes.

Recently, another isoform of the MRP family, MRP3, was cloned from human liver (33, 34, 53) and localized to the basolateral hepatocyte membrane (33, 34). Immunoblot analysis (Fig. 2) and reverse transcription PCR analysis (Fig. 3) revealed that MRP3 is expressed in Hep G2 cells. The lateral localization of MRP3 was confirmed by double-label immunofluorescence microscopy with the FDS antibody, directed against MRP3, and by simultaneous detection of the apically localized ectoenzyme DPPIV (Fig. 1).

Significant amounts of MRP1 mRNA were not detected in normal human liver (8, 32). However, Hep G2 cells showed expression of MRP1 mRNA (Fig. 3). On the protein level, MRP1 was not detectable by immunoblotting (Fig. 2); however, a fraction of Hep G2 cells showed weak plasma membrane staining with the QCRL-1 antibody by immunofluorescence. This is consistent with the very low expression of MRP1 described earlier for Hep G2 cells (23). MRP1 expression in Hep G2 cells was also observed by Roelofsen et al. (45). Studies are currently in progress to determine which culture conditions, such as cell density and cell passage number, favor an expression of MRP1 in Hep G2 cells.

MRP2-mediated transport of fluo 3 and carboxyfluorescein. In a previous study, we identified fluo 3 as a substrate of relatively high affinity for rat MRP2 by measurement of its ATP-dependent transport into inside-out membrane vesicles from MRP2-containing hepatocyte canalicular membrane vesicles and by its secretion into apical vacuoles of rat hepatoma WIF-B cells (36). Fluo 3 was mainly sequestered into vacuolar structures of Hep G2 cells, which were identified as apical vacuoles by subsequent immunofluorescence analysis using an antibody specifically recognizing human MRP2 (Fig. 4). Presence of human MRP2 in the apical membrane of Hep G2 cells (Fig. 1) and similar substrate specificities of rat and human MRP2 (9) suggest that fluo 3 is also a substrate for human MRP2.

In addition to MRP2, the apical membrane of Hep G2 cells also contains P-glycoprotein transporters (45), which include the BSEP mediating the ATP-dependent transport of the bile salt cholyltaurine (12) and the protein excreting chemotherapeutic drugs such as daunorubicin (MDR1 P-glycoprotein; Ref. 25). All three ATP-dependent transporters are inhibited, although to different extents, by cyclosporin A and its nonimmunosuppressive analog PSC833 (4). Fluo 3 secretion into apical vacuoles of Hep G2 cells was inhibited by cyclosporin A; however, a concentration of 10 µM was required for 50% inhibition (Fig. 6) compared with 3.5 µM for rat MRP2-mediated fluo 3 secretion into apical vacuoles of WIF-B cells (36). Because cyclosporin A also strongly inhibits daunorubicin and cholyltaurine transport (4), fluo 3 secretion into apical vacuoles had to be discriminated from P-glycoprotein-mediated transport. We therefore used PSC833, which is a strong inhibitor of P-glycoprotein-mediated daunorubicin and bile salt transport but a weak inhibitor of MRP2-mediated leukotriene C4 transport in rat canalicular membrane vesicles (4). In the presence of 10 µM PSC833, only a weak inhibition of fluo 3 secretion into apical vacuoles of Hep G2 cells was observed (Fig. 6). In addition, LY-335979, which selectively inhibits MDR1 P-glycoprotein-mediated transport with an inhibition constant of 60 nM (10) but not MRP1- or MRP2-mediated transport (11), had no effect on fluo 3 secretion (Fig. 6). A similar inhibition pattern was observed for fluo 3 secretion into apical vacuoles of rat hepatoma WIF-B cells (36). Inhibition of fluo 3 secretion by cyclosporin A, but not by a selective MDR1 P-glycoprotein inhibitor, establishes further that fluo 3 is transported into the apical vacuoles of Hep G2 cells by human MRP2 and not by MDR1 P-glycoprotein.

Intensity of fluo 3 fluorescence depends on Ca2+ concentration (26, 35). Therefore, the decrease in fluo 3 fluorescence after incubation with inhibitors may reflect changes in the Ca2+ concentration inside the apical vacuoles rather than inhibition of fluo 3 secretion into the apical vacuoles. However, studies with isolated membrane vesicles demonstrated that MRP2-mediated transport of fluo 3 is indeed inhibited by cyclosporin A (36). In addition, we used the fluorescent CF, which is not intracellularly conjugated to glutathione (2). CF was recently described as being effluxed from MRP1-expressing tumor cells (54). CF accumulated in apical vacuoles of Hep G2 cells (Fig. 4). CF secretion was inhibited by cyclosporin A, showing a similar pattern to that observed for inhibition of fluo 3 secretion (Fig. 6). Because of similar substrate specificities of MRP1 and MRP2 (29), CF is likely to be transported by MRP2 across the apical membrane of Hep G2 cells. Glutathionylmethylfluorescein, which has been used as a substrate for rat MRP2 (44), was also transported into apical vacuoles of Hep G2 cells (45).

Transport of rhodamine 123 and fluorescent bile salt derivatives. The fluorescent membrane-permeable compound rhodamine 123 has been used for staining of mitochondria in live cells (24) as well as to detect MDR1 P-glycoprotein transport activity (51, 55). In Hep G2 cells, rhodamine 123 accumulated, as expected, in mitochondria and, in addition, in apical vacuoles (Fig. 5). Secretion into apical vacuoles, but not into mitochondria, was inhibited by the selective MDR1 P-glycoprotein inhibitor LY-335979 (Fig. 5), which is consistent with the presence of MDR1 P-glycoprotein in the apical membrane of Hep G2 cells.

Two fluorescent bile salt derivatives were used to investigate whether the apical membrane of Hep G2 cells contains BSEP. CGamF, a fluorescent derivative of the bile salt cholylglycine, was recently synthesized (16) and shown to be taken up into rat hepatocytes and secreted into bile (15, 16). In Hep G2 cells, CGamF was secreted into the apical vacuoles (Fig. 5). Transport of CGamF across the apical membrane of Hep G2 cells is likely to be mediated by the BSEP, also known as sister of P-glycoprotein (7, 12), rather than by MRP2. Cyclosporin A differentially inhibits distinct ATP-dependent transporters in the canalicular membrane of hepatocytes (4). A 40% inhibition of CGamF secretion was observed at a concentration of 0.5 µM cyclosporin A, whereas a 10-fold higher concentration was needed to cause a similar reduction of MRP2-mediated fluo 3 secretion (Fig. 6). This finding supports that CGamF transport is mediated by BSEP, whose presence in Hep G2 cells will require detection with a BSEP-specific antibody.

In contrast to CGamF, UDC-L-NBD was mainly sequestered into intracellular vesicles of Hep G2 cells, in addition to its secretion into the apical vacuoles (Fig. 5). Part of these intracellular structures also accumulated the acidophilic LysoTracker Red. The fluorescent cation daunorubicin was recently shown to be accumulated in intracellular acidic organelles (13). Sequestration of UDC-L-NBD was also observed in the perfused rat liver, and the smooth endoplasmic reticulum was suggested as a candidate intracellular structure (15). Transport of UDC-L-NBD across the apical membrane of Hep G2 cells is most likely not mediated by MRP2, because the fluorescent bile salt is well transported in MRP2-deficient mutant rats (16). The candidate transport protein may be BSEP or another yet-unidentified transporter in the apical membrane of Hep G2 cells.

The stronger inhibition by cyclosporin A of CGamF and UDC-L-NBD secretion relative to fluo 3 and CF secretion indicates that the amphiphilic anions fluo 3 and CF are transported by another membrane protein than the fluorescent bile salts. However, transport studies with isolated membrane vesicles containing recombinant BSEP are required to directly show transport of both fluorescent bile salts and specific inhibition by cyclosporin A and other inhibitors.

In conclusion, human MRP2 is expressed in the apical membrane of polarized Hep G2 cells and is functionally active in transporting fluorescent amphiphilic anions such as fluo 3 and CF. This cell system proves to be useful to study the function of human MRP2 in a cultured living cell line and to examine the action of inhibitors of MRP2-mediated transport.


    ACKNOWLEDGEMENTS

We thank Dr. Jörg König for the PCR primers and the FDS antibody (34), Dr. Gabriele Jedlitschky for immunoblotting of MRP3-expressing MDCK cells, Dr. Yunhai Cui for providing MRP3-expressing MDCK cells (34), and Dr. Roger G. Deeley and Dr. Susan P. C. Cole for the MRP1-transfected HeLa T5 cells and the QCRL-1 antibody.


    FOOTNOTES

This work was supported in part by grants from the Deutsche Forschungsgemeinschaft through SFB 352/B3 and SFB 601/A2, by the Tumorzentrum Heidelberg/Mannheim, and by the Forschungsschwerpunkt Transplantation, Heidelberg. Work at UCSD was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-21506 and a Grant-in-Aid from the Falk Foundation, Freiburg, Germany.

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: A. Nies, Division of Tumor Biochemistry, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany (E-mail: a.nies{at}dkfz-heidelberg.de).

Received 23 July 1999; accepted in final form 17 November 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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