pharmaceutics

Article mRNA Expression and Activity of Transporters in Human Hepatoma HepaRG Cells

Abdullah Mayati 1, Amélie Moreau 2, Elodie Jouan 1, Marie Febvre-James 1, Claire Denizot 2, Yannick Parmentier 2 and Olivier Fardel 1,3,*

1 Univ Rennes, Inserm, EHESP, IRSET (Institut de Recherche en Santé, Environnement et Travail) - UMR_S 1085, F-35000 Rennes, France; [email protected] (A.M.); [email protected] (E.J.); [email protected] (M.F.-J.) 2 Centre de Pharmacocinétique, Technologie Servier, F-45000 Orléans, France; amé[email protected] (A.M.); [email protected] (C.D.); [email protected] (Y.P.) 3 Pôle Biologie, Centre Hospitalier Universitaire, F-35033 Rennes, France * Correspondence: [email protected]; Tel.: +33-223-23-4880

 Received: 10 September 2018; Accepted: 16 November 2018; Published: 21 November 2018 

Abstract: The HepaRG cell line is a highly differentiated human hepatoma cell line, displaying the expression of various drug transporters. However, functional expression of nucleoside transporters remains poorly characterized in HepaRG cells, although these transporters play a key role in hepatic uptake of antiviral and anticancer drugs. The present study was, therefore, designed to characterize the expression, activity and regulation of equilibrative (ENT) and concentrative (CNT) nucleoside transporter isoforms in differentiated HepaRG cells. These cells were found to exhibit a profile of nucleoside transporter mRNAs similar to that found in human hepatocytes, i.e., notable expression of ENT1, ENT2 and CNT1, with very low or no expression of CNT2 and CNT3. ENT1 activity was, next, demonstrated to be the main transport activity present in HepaRG cells, like in cultured human hepatocytes. Various physiological factors, such as protein kinase C (PKC) activation or treatment by inflammatory cytokines or hepatocyte growth factor (HGF), were additionally found to regulate expression of ENT1, ENT2 and CNT1; PKC activation and HGF notably concomitantly induced mRNA expression and activity of ENT1 in HepaRG cells. Overall, these data suggest that HepaRG cells may be useful for analyzing cellular pharmacokinetics of nucleoside-like drugs in human hepatic cells, especially of those handled by ENT1.

Keywords: ENT1; nucleoside transporter; HepaRG cells; hepatocytes; uridine

1. Introduction The hepatic cell line HepaRG is an original human hepatoma cell line, which expresses various liver-specific functions when cultured in appropriate conditions, i.e., in the presence of 2% (vol/vol) dimethyl sulfoxide (DMSO) [1]. These hepatoma cells have, consequently, been proposed as surrogates for the use of primary human hepatocytes, especially for xenobiotic metabolism and toxicity studies [2,3]. This assertion is notably validated by the fact that HepaRG cells display functional activities of main drug hepatic detoxifying proteins, including drug metabolizing like cytochromes P-450 [4], but also drug transporters [5], whose major role in hepatobiliary elimination of xenobiotics is now well-established. Importantly, HepaRG cells exhibit polarized expression of drug transporters, with expression of solute carrier (SLC) transporters like organic cation transporter 1 (OCT1/SLC22A1) and organic anion transporting polypeptides (OATPs/SLCOs) 1B1 (OATP1B1/SLCO1B1) and 2B1 (OATP2B1/SLCO2B1) at their sinusoidal pole, and expression

Pharmaceutics 2018, 10, 246; doi:10.3390/pharmaceutics10040246 www.mdpi.com/journal/pharmaceutics Pharmaceutics 2018, 10, 246 2 of 18 of drug efflux ATP-binding cassette (ABC) transporters like P-glycoprotein (ABCB1) and multidrug resistance-associated protein 2 (MRP2/ABCC2) at their canalicular pole [6]. Expression of drug transporters in HepaRG cells thus mimicks that displayed by primary human hepatocytes [7]. Concentrative and equilibrative nucleoside transporters (CNT/SLC28 and ENT/SLC29) are two families of transmembrane proteins that mediate transport of hydrophilic across cell membranes [8,9]. The CNT family includes three members, i.e., CNT1 (SLC28A1), CNT2 (SLC28A2) and CNT3 (SLC28A3), which are sodium-dependent symporters, that mediate unidirectional transport of nucleosides into cells [10]. The ENT family comprises four members, i.e., ENT1 (SLC29A1), ENT2 (SLC29A2), ENT3 (SLC29A3) and ENT4 (SLC29A4)[11]. ENT1 and ENT2 mediate passive bidirectional transport of purine and pyrimidine nucleosides down a concentration gradient across the plasma membrane; ENT3 transports nucleosides across intracellular membranes, notably those of lysosomes [12], whereas ENT4, also known as plasma membrane (PMAT), mainly functions as a polyspecific organic cation transporter [13], handling only among nucleosides [14]. Thereby, only ENT1 and ENT2 among ENTs can be considered as prototypical plasma membrane nucleoside transporters. Unlike the broad specificity of ENT1/2 and CNT3, CNT1 and CNT2 primarily handle pyrimidine and purine nucleosides, respectively [15]. Uridine is an ubiquitous substrate for CNTs and ENT1/2 [16]. Both CNTs and ENTs are likely to contribute to the cellular pharmacokinetics and, in this way, to the pharmacological activity of various nucleoside analogs used in antiviral or anticancer therapy [17]. Nucleoside transporters are expressed in hepatocytes, notably ENT1, ENT2 and CNT1 [18,19]. They have been implicated, especially ENT1, in the hepatic uptake of the antiviral agent ribavirin [20,21], as well as in that of the anticancer drug gemcitabine [22]. However, whether HepaRG cells exhibit functional expression and regulation of nucleoside transporters remains unknown. Nevertheless, this point is worthy of interest, owing to (i) the established role played by nucleoside transporters in pharmacokinetics and toxicity of various drugs and xenobiotics; and (ii) the growing use of HepaRG cells as surrogates for human hepatocytes in pharmacological and toxicological studies [2,3]. Therefore, the present study was designed to characterize the expression of plasma membrane nucleoside transporters, i.e., ENT1, ENT2, CNT1, CNT2 and CNT3, in HepaRG cells, as well as the putative regulation of some of them by factors known to modulate drug transporter expression in human hepatic cells, like protein kinase C (PKC) activation [23] or inflammatory cytokine treatment [24].

2. Materials and Methods

2.1. Chemicals and Reagents S-(4-Nitrobenzyl)-6-thioinosine (NBMPR), phorbol myristate acetate (PMA), lipopolysaccharide (LPS) (Escherichia coli O55:B5) and bovine insulin were provided by Sigma-Aldrich (Saint-Quentin Fallavier, France). Ruxolitinib was from Selleckchem (Houston, TX, USA), whereas the PKC inhibitors GF 109203X and Gö 6976 were from Calbiochem (La Jolla, CA, USA). Recombinant human hepatocyte growth factor (HGF), tumor necrosis factor (TNF) α, interleukin (IL) 6 and IL1β were purchased from R&D Systems (Minneapolis, MN, USA). [5-3H]-uridine (specific activity = 20.4 Ci/mmol) was from PerkinElmer (Courtaboeuf, France). All other reagents were commercial products of the highest purity available.

2.2. Cell Culture HepaRG cells were routinely plated at low density (27,000 cells/cm2) and cultured in Williams’ E medium supplemented with 10% (vol/vol) fetal calf serum, 20 µg/mL streptomycin, 20 IU/mL penicillin, 2 mM glutamine, 5 µg/mL bovine insulin, and 50 µM hydrocortisone hemisuccinate. After two weeks, cells were trypsinated for passaging or cultured for additional two weeks in the Pharmaceutics 2018, 10, 246 3 of 18 same medium added with 2% (vol/vol) DMSO, in order to obtain differentiated hepatocytes-like cells, as previously described [1]. Freshly isolated human hepatocytes were obtained from the Biological Resource Center BB-0033-00056 (University Hospital, Rennes, France), which has obtained the authorization No DC-2008-630 from the French Ministry of Health to collect hepatic resections from the digestive surgery department and then to isolate and deliver the hepatocytes. All liver fragment donors were adult and provided a written informed consent to participate in the study. All experimental procedures complied with French laws and regulations; they were approved by the National Ethics Committee from INSERM (IRB00003888). Upon delivery, human hepatocytes were seeded on plastic dishes at a high density (250,000 cells/cm2) in Williams’ E medium, supplemented with 10% (vol/vol) fetal calf serum, 5 µg/mL bovine insulin, 20 IU/mL penicillin, 20 µg/mL streptomycin, and 2 mM glutamine. After a 24 h-seeding culture period, the medium was discarded and primary hepatocytes were next cultured for 6 days in the DMSO-containing HepaRG cell-differentiating medium described above, as previously described [7]. The human hepatoma cell line HuH-7 was cultured in Dulbecco’s modified Eagle medium (DMEM, Life Technologies), supplemented with 10% (vol/vol) fetal calf serum, 20 IU/mL penicillin and 20 µg/mL streptomycin, as previously described [25]. Human macrophages were obtained from peripheral blood monocytes as previously reported [26]. Briefly, peripheral blood mononuclear cells were first isolated from blood buffy coats of healthy donors through Ficoll gradient centrifugation. After a 1 h adhesion step, the cells were cultured for 6 days in RPMI 1640 medium, supplemented with 10% (vol/vol) fetal calf serum, 2 mM glutamine, 20 IU/mL penicillin and 20 µg/mL streptomycin, in the presence of 400 IU/mL GM-CSF. Nearly-haploid HAP1 cells and ENT1-knockout HAP1 (HAP1 ENT14) cells, edited by CRISPR/Cas9 to contain a 14 bp deletion in a coding exon of ENT1, were obtained from Horizon Discovery (Cambridge, UK). They were routinely cultured in Iscove’s modified Dulbecco’s medium (IMDM) (Thermo Fisher Scientific) supplemented with 10% (vol/vol) fetal calf serum, 20 IU/mL penicillin and 20 µg/mL streptomycin.

2.3. RNA Isolation and Analysis Total RNAs were extracted from cells using the TRI reagent (Sigma-Aldrich), and were then reverse-transcribed to cDNA using the reverse-transcription (RT) kit from Applied Biosystems (Foster City, CA, USA). Quantitative polymerase chain reaction (qPCR) assays were next performed using the fluorescent dye SYBR Green methodology and a CFX384 real-time PCR detection system (Bio-Rad, Marnes-la-Coquette, France), as previously described [6]. Gene primers were: CNT1 sense, AGGTCCTGCCCATCATTGTC, CNT1 anti-sense, CAAGTAGGGCCGGATCAGTA, CNT2 sense, AATGGGTGTTTGCAGGAGTC, CNT2 anti-sense, GAAGACCTAGGCCCGAAAAC, CNT3 sense, GACTCACATCCATGGCTCCT, CNT3 antisense, TTCCAGGGAAAGTGGAGTTG, ENT1 sense, CCTGGCTTTCTCTGTCTGCT, ENT1 anti-sense, AGTAACGTTCCCAGGTGCTG, ENT2 sense, CCCTGGATCTTGACCTGGAG, ENT2 anti-sense, GGTTTTCCTGGCTTCTGGG, 18S rRNA sense, CGCCGCTAGAGGTGAAATTC and 18S rRNA anti-sense, TTGGCAAATGCTTTCGCT. The specificity of each gene amplification was verified at the end of qPCR reactions through analysis of dissociation curves of the PCR products. Amplification curves were analyzed with the CFX Manager software (Bio-Rad), using the comparative cycle threshold method. Relative quantification of the steady-state target mRNA levels was calculated after normalization of the total amount of cDNA tested to the 18S rRNA endogenous reference, using the 2(−∆∆Ct) method. Even if the use of several reference genes has been preconized [27], that of the 18S rRNA as a unique reference gene has been previously successfully retained for investigating transporter mRNA regulation in response to PMA or cytokines in HepaRG cells [23,28]. Indeed, the amount of 18S rRNA is presumed to remain constant, whatever the cell culture conditions. In agreement with this assertion, the amount of 18S rRNA in proliferating and differentiated HepaG cells did not vary, i.e., the Ct numbers determined from qPCR assays were Pharmaceutics 2018, 10, 246 4 of 18 similar in these cells (Figure S1). Data of qPCR experiments were finally commonly expressed in arbitrary units relatively to 18S rRNA or as fold change comparatively to control untreated cells, as previously reported [29].

2.4. Immunofluorescence Assays Cells were fixed with ice-cold acetone for 10 min, washed with phosphate-buffered saline (PBS) and incubated in a 4% (weight/vol) bovine serum albumin/PBS blocking solution for 1 h at room temperature. Cells were next incubated overnight with 1:50 (vol/vol) working dilutions (4 µg/mL) of primary monoclonal mouse antibodies targeting CNT1 (clone G-1), ENT1 (clone F-12) or ENT2 (clone A-8), provided by Santa Cruz Biotechnology (Dallas, TX, USA). Cells were then washed twice with PBS, before adding secondary antibody directed against mouse IgG and coupled to Alexa Fluor 488 (Cell Signaling, Leiden, The Netherlands), for 45 min. In some experiments, additional labelling of F-actin with Alexa Fluor 555-coupled phalloidin (Ozyme, Montigny-le-Bretonneux, France) was performed for 45 min. Nuclei were next stained by 40,6-diamidino-2-phenylindole (DAPI) for 10 min. Cells were finally washed three times with PBS and images of immuno-labeling were acquired with a confocal fluorescence microscope LEICA DMI 6000 CS (Leica Microsystemes SAS, Nanterre, France).

2.5. Transport Assays For transport assays, uridine was used as a specific and common substrate for CNT1, CNT2, CNT3, ENT1 and ENT2 [9]. Uridine is also a substrate for ENT3, but the intracellular location of this transporter discards any role in initial cellular uptake of uridine [12], whereas ENT4 fails to transport uridine [9]. To discriminate among CNT and ENT activities, we used the differential sensitivity of these transporters to sodium withdrawal and to inhibitors such as NBMPR (which inhibits ENT1 when used at 100 nM and ENT1 and ENT2 when used at 100 µM, but not CNTs) [30], thymidine (blocking CNT1 and CNT3) [19] and (blocking CNT2 and CNT3) [31] (Table1).

Table 1. Differential sensitivity of nucleoside transporters to inhibitors.

Nucleoside Transporter Inhibitor CNT1 CNT2 CNT3 ENT1 ENT2 Sodium whithdrawal + + + − − S-(4-Nitrobenzyl)-6-thioinosine (NBMPR) − − − + − (100 nM) NBMPR (100 µM) − − − + + Inosine (200 µM) − + + Not relevant Not relevant Thymidine (200 µM) + − + Not relevant Not relevant + inhibition; − no inhibition.

Cells were incubated with 24.5 nM [3H]-uridine for 5 min at 37 ◦C, in a well-defined transport assay buffer consisting of 5.3 mM KCl, 1.1 mM KH2PO4, 0.8 mm MgSO4, 1.8 mM CaCl2, 11 mM d-glucose, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and 136 mM N-methyl-glucamine (sodium-free buffer) or 136 mM NaCl (sodium-containing buffer) and adjusted to pH 7.4, in the presence or absence of NBMPR (used at 100 nM or 100 µM), 200 µM inosine or 200 µM thymidine. After washing in PBS, cells were lysed, and accumulation of radiolabeled uridine was determined through scintillation counting and normalized to total protein content, determined by the Bradford method. Nucleoside transporter activities related to uridine uptake were next determined using the following equations: ENTTotal activity = U-Na − U-Na/+ 100 µM NBMPR (1)

ENT1 activity = U-Na − U-Na/+ 100 nM NBMPR (2) Pharmaceutics 2018, 10, 246 5 of 18

ENT2 activity = ENTTotal activity − ENT1 activity (3)

CNTTotal activity = U+Na/+ 100 µM NBMPR − U-Na/+ 100 µM NBMPR (4)

CNT1 activity = U+Na/+ 100 µM NBMPR/+ 200 µM Inosine − U-Na/+ 100 µM NBMPR (5)

CNT2 activity = U+Na/+ 100 µM NBMPR/+ 200 µM Thymidine − U-Na/+ 100 µM NBMPR (6)

CNT3 activity = CNTTotal activity − (CNT1 activity + CNT2 activity) (7) with U-Na = uridine uptake in the absence of sodium, U-Na/+ 100 µM NBMPR = uridine uptake in the absence of sodium and presence of 100 µM NBMPR (this corresponds to passive uptake of uridine, when CNTs and ENTs are not functional), U+Na/+ 100 µM NBMPR = uridine uptake in the presence of sodium and 100 µM NBMPR, U+Na/+ 100 µM NBMPR/+ 200 µM Inosine = uridine uptake in the presence of sodium, 100 µM NBMPR and 200 µM inosine and U+Na/+ 100 µM NBMPR/+ 200 µM Thymidine = uridine uptake in the presence of sodium, 100 µM NBMPR and 200 µM thymidine.

2.6. Statistical Analysis Quantitative data were routinely expressed as means ± standard error of the mean (S.E.M.) of at least three independent assays. Statistical analysis was performed using the Student’s t test or by analysis of variance (ANOVA) followed by Dunnett’s or Tukey’s post hoc tests. The criterion of significance was p < 0.05.

3. Results

3.1. Expression of Nucleoside Transporters in HepaRG cells Expression of nucleoside transporter mRNAs was determined by RT-qPCR in proliferating HepaRG cells (cells cultured for 3 days after initial seeding, without DMSO) and in differentiated HepaRG cells (cells cultured for 15 days after seeding without DMSO, followed by an additional 15 day-culture period in the presence of DMSO), as well as in freshly isolated hepatocytes and primary cultured human hepatocytes. As indicated in Figure1, ENT1 and ENT2 were expressed at substantial levels in freshly isolated and primary hepatocytes, and in proliferating and differentiated HepaRG cells. ENT1 mRNA expression in differentiated HepaRG cells was, however, significantly lower than those found in freshly isolated and cultured hepatocytes; it was also lower than that found in proliferating HepaRG cells, even if statistical significance was not reached. ENT2 mRNA expression was significantly higher in differentiated HepaRG cells than in freshly isolated hepatocytes and proliferating HepaRG cells (Figure1). CNT1 mRNAs were expressed at substantial level in freshly isolated and primary cultured hepatocytes, as well as in differentiated HepaRG cells, but were barely detected in proliferating HepaRG cells. CNT1 mRNA level in differentiated HepaRG cells was similar to that found in primary human hepatocytes and slightly, but significantly, lower than that observed in freshly isolated human hepatocytes. Both CNT2 and CNT3 were not expressed, or only at a very low level, in hepatocytes and proliferating and differentiated HepaRG cells (Figure1). Pharmaceutics 2018, 10, 246 6 of 18 Pharmaceutics 2018, 10, x FOR PEER REVIEW 6 of 18 Pharmaceutics 2018, 10, x FOR PEER REVIEW 6 of 18

Freshly isolated hepatocytes FreshlyPrimary isolated cultured hepatocytes human hepatocytes PrimaryProliferating cultured HepaRG human cellshepatocytes ProliferatingDifferentiated HepaRG HepaRG cells cells Differentiated HepaRG cells

* 10 * 10 * * 8 8 * * 6 * 6 * * * 4 * 4 * (arbitrary units) mRNA expression (arbitrary units)

mRNA expression 2 2

0 0 CNT1 CNT2 CNT3 ENT1 ENT2 CNT1 CNT2 CNT3 ENT1 ENT2

FigureFigure 1. Expression 1. Expression of nucleosideof nucleoside transporter transporter mRNAs mRNAs in in proliferating proliferating andand differentiated HepaRG HepaRG Figure 1. Expression of nucleoside transporter mRNAs in proliferating and differentiated HepaRG cellscells and and in freshly in freshly isolated isolated and and primary primary cultured cultured human human hepatocytes. hepatocytes. Expression of of CNT1, CNT1, CNT2, CNT2, cells and in freshly isolated and primary cultured human hepatocytes. Expression of CNT1, CNT2, CNT3,CNT3, ENT1 ENT1 and and ENT2 ENT2 mRNAs mRNAs was was analyzed analyzed by by reverse-transcription reverse-transcription quantitativequantitative polymerase polymerase chain chain CNT3, ENT1 and ENT2 mRNAs was analyzed by reverse-transcription quantitative polymerase chain reactionreaction (RT-qPCR). (RT-qPCR). Data Data are are expressed expressed in arbitrary in arbitrar unitsy units and and are are the the means means± S.E.M.± S.E.M. of of values values from from at reaction (RT-qPCR). Data are expressed in arbitrary units and are the means ± S.E.M. of values from leastat seven least independentseven independent human human hepatocyte hepatocyte populations populations and five and independent five independent HepaRG HepaRG cell cultures. cell at least seven independent human hepatocyte populations and five independent HepaRG cell *, p

Figure 2. Immunolocalization of ENT1, ENT2 and CNT1 in proliferating HepaRG cells. ENT1, ENT2 Figure 2. Immunolocalization of ENT1, ENT2 and CNT1 in proliferating HepaRG cells. ENT1, ENT2 Figureand 2.CNT1Immunolocalization were immunolocalized of ENT1, in proliferatin ENT2 andg CNT1HepaRG in cells, proliferating as described HepaRG in Materials cells. ENT1, and and CNT1 were immunolocalized in proliferating HepaRG cells, as described in Materials and ENT2Methods. and CNT1 Nucleoside were immunolocalized transporter immunolabeling in proliferating appears HepaRG as green cells, fluorescence as described on inmicroscopy Materials Methods. Nucleoside transporter immunolabeling appears as green fluorescence on microscopy andpictures, Methods. whereas Nucleoside 4′,6-diamidino-2-phenylindole transporter immunolabeling (DAPI)-stained appears as green nuclei fluorescence are blue. White on microscopy arrows pictures, whereas0 4′,6-diamidino-2-phenylindole (DAPI)-stained nuclei are blue. White arrows pictures,indicate whereas membrane 4 ,6-diamidino-2-phenylindole labelling. Data are representati (DAPI)-stainedve of three independent nuclei are blue. assays. White White arrows bar = indicate50 µm. indicate membrane labelling. Data are representative of three independent assays. White bar = 50 µm. membrane labelling. Data are representative of three independent assays. White bar = 50 µm.

Pharmaceutics 2018, 10, 246 7 of 18 Pharmaceutics 2018, 10, x FOR PEER REVIEW 7 of 18

FigureFigure 3. 3.Immunolocalization Immunolocalization of of ENT1, ENT1, ENT2 ENT2 and and CN CNT1T1 in differentiated in differentiated HepaRG HepaRG cells. (A cells.) ENT1 (A ) ENT1 andand ENT2 ENT2 were were immunolocalizedimmunolocalized in in differentiated differentiated HepaRG HepaRG cells, cells, as described as described in Materials in Materials and and Methods.Methods. NucleosideNucleoside transporter transporter immunolabeling immunolabeling appears appears as green as green fluorescence fluorescence on microscopy on microscopy pictures, whereas DAPI-stained nuclei are blue. (B) CNT1 and phalloidin-labeled F-actin were co- pictures, whereas DAPI-stained nuclei are blue. (B) CNT1 and phalloidin-labeled F-actin were localized in differentiated HepaRG cells. White arrows indicate pericanalicular labeling of CNT1 and co-localized in differentiated HepaRG cells. White arrows indicate pericanalicular labeling of CNT1 F-actin. (A,B) Data are representative of three independent assays. White bar = 50 µm. and F-actin. (A,B) Data are representative of three independent assays. White bar = 50 µm. 3.2. Nucleoside Transporter Activities in HepaRG Cells 3.2. Nucleoside Transporter Activities in HepaRG Cells Analysis of nucleoside transporter activities was based on the measurement of uridine uptake in Analysisthe absence of or nucleoside presence of transporter sodium and/or activities various was inhibitors. based onThese the methods measurement highlighted of uridine CNT1, uptake in theENT1 absence and ENT2 or presenceactivities in of pr sodiumimary human and/or hepatocytes, various inhibitors. whereas CNT2 These and methods CNT3 activities highlighted were CNT1, ENT1not detected and ENT2 (Figure activities 4). Such in primarya profile humanof nucleoside hepatocytes, transporter whereas activities CNT2 fullyand agrees CNT3 with activities that of were notnucleoside detected transporter (Figure4). mRNA Such aex profilepression, of i.e., nucleoside expression transporter of CNT1, ENT1 activities and ENT2 fully agreesmRNAs with in that ofprimary nucleoside human transporter hepatocytes mRNA (Figure expression, 1). However, i.e., it is expression noteworthy ofthat CNT1, total uridine ENT1 uptake and ENT2 in human mRNAs in primaryhepatocytes human in hepatocytesthe presence of (Figure sodium1). (both However, CNTs itand is noteworthyENTs are functional) that total is not uridine higher uptake than that in human hepatocytesin the absence in the of sodium presence (only of sodiumENTs are (both functional). CNTs andThis ENTsmay be are due functional) to the fact isthat not CNT1 higher activity than that in theis absence low when of sodiumcompared (only to ENTsENT1 areactivity functional). (Figure 4) This and/or may to be the due reduced to the fact number that CNT1(four) activityof is independent hepatocyte populations analyzed in the functional assay. Alternatively, ENT1 is an low when compared to ENT1 activity (Figure4) and/or to the reduced number (four) of independent equilibrative bidirectional transporter [21] and it may be hypothesized that, when CNT1 activity is hepatocytefunctional populations(in the presence analyzed of sodium), in the high functional uridine intracellular assay. Alternatively, concentration ENT1 may isbe an reached, equilibrative bidirectionalwhich may restrain transporter ENT1 [activity.21] and it may be hypothesized that, when CNT1 activity is functional (in the presence of sodium), high uridine intracellular concentration may be reached, which may restrain ENT1 activity.

Pharmaceutics 2018, 10, 246 8 of 18 Pharmaceutics 2018, 10, x FOR PEER REVIEW 8 of 18

Figure 4. Uridine uptake in primary cultured human hepatocytes. Primary human hepatocytes were Figure 4. Uridine uptake in primary cultured human hepatocytes. Primary human hepatocytes were incubated with 24.5 nM [3H]-uridine for 5 min at 37 ◦C, in the presence or absence of sodium and in incubated with 24.5 nM [3H]-uridine for 5 min at 37 °C, in the presence or absence of sodium and in the presence or absence of the ENT inhibitor NBMPR (used at 100 nM or 100 µM) or the CNTs inhibitor the presence or absence of the ENT inhibitor NBMPR (used at 100 nM or 100 µM) or the CNTs thymidine or inosine. Uridine accumulation was next determined by scintillation counting. Data are inhibitor thymidine or inosine. Uridine accumulation was next determined by scintillation counting. expressed as pmol uridine/mg protein and are the means ± S.E.M. of values from four independent Data are expressed as pmol uridine/mg protein and are the means ± S.E.M. of values from four human hepatocyte populations. Activities of CNT1, ENTs, ENT1 and ENT2, defined by equations independent human hepatocyte populations. Activities of CNT1, ENTs, ENT1 and ENT2, defined by described in Materials and Methods, are indicated by double arrows on the graphs. equations described in Materials and Methods, are indicated by double arrows on the graphs. To further confirm the relevance of the uridine uptake-based assay for discriminating among nucleosideTo further transporter confirm activities,the relevance we of performed the uridin ite inuptake-based various cell assay types for differentially discriminating expressing among nucleoside transporters.transporter activities, Hepatoma we HuH-7 performed cells, whichit in various exhibited cell mainly types ENT2differentially mRNA expressing expression nucleoside(Figure S2A, transporters. left panel), displayedHepatoma marked HuH-7 ENT2cells, activitywhich exhibited (Figure S2A, mainly right ENT2 panel). mRNA Primary expression human (Figuremacrophages, S2A, left which panel), highly displayed expressed marked CNT3 ENT2 (Figure activity S2B, left (Figure panel)), S2A, concomitantly right panel). exhibited Primary markedhuman macrophages,CNT3 activity which (Figure highly S2B, rightexpressed panel). CNT3 Finally, (Figure HAP1 S2B, cells left displayed panel)), membraneconcomitantly expression exhibited of markedENT1 (Figure CNT3 S3A)activity and (Figure ENT1 S2B, activity right (Figure panel). S3B); Finally, by contrast, HAP1 cells HAP1 displayed ENT14 membranecells failed expression to exhibit △ ofmembrane ENT1 (Figure expression S3A) and of ENT1 ENT1 (Figure activity S3A) (Figure and ENT1S3B); by activity contrast, (Figure HAP1 S3B). ENT1 Taken cells together, failed theseto exhibit data membranesupport the expression conclusion of thatENT1 the (Figure uridine S3A) uptake-based and ENT1 activity assay permits (Figure to S3B). detect Taken specific together, activity these of datanucleoside support transporters the conclusion when that they the are uridine substantially uptake-b expressed.ased assay Consequently,permits to detect we specific applied activity this assay of nucleosideto proliferating transporters and differentiated when they are HepaRG substantially cells. CNT expressed. total activity, Consequently, i.e., the we uptake applied of uridinethis assay in tothe proliferating presence of and sodium differentiated and 100 µ HepaRGM NBMPR cells. minus CNT that total in activity, the absence i.e., the of sodiumuptake of and uridine presence in the of presence100 µM NBMPR, of sodium was and not 100 detected µM NBMPR or only minus at a very that low in levelthe absence in both of proliferating sodium and and presence differentiated of 100 µMHepaRG NBMPR, cells was (Figure not5 );detected consequently, or only the at effectsa very oflow the level CNT in inhibitors both proliferating thymidine and and differentiated inosine were HepaRGnot analyzed. cells By(Figure contrast, 5); consequently, ENT1 activity the was effects detected of the in bothCNT proliferating inhibitors thymidine and differentiated and inosine HepaRG were notcells analyzed. and corresponded By contrast, to nearly ENT1 total activity ENT activity, was dete indicatingcted in thatboth ENT2 proliferating activity was and barely differentiated detectable HepaRG(Figure5). cells and corresponded to nearly total ENT activity, indicating that ENT2 activity was barely detectable (Figure 5).

Pharmaceutics 2018, 10, x FOR PEER REVIEW 9 of 18 Pharmaceutics 2018, 10, 246 9 of 18 Pharmaceutics 2018, 10, x FOR PEER REVIEW 9 of 18

Figure 5. Uridine uptake in proliferating (A) and differentiated (B) HepaRG cells. Proliferating and

3 differentiatedFigure 5. Uridine HepaRG uptake cells in proliferatingwere incubated (A) with and differentiated24.5 nM [ H]-uridine (B) HepaRG for 5 cells. min Proliferatingat 37 °C, in the and Figure 5. Uridine uptake in proliferating (A) and differentiated (B) HepaRG cells. Proliferating and presencedifferentiated or absence HepaRG of sodium cells were and incubated in the presence with 24.5 or nM absence [3H]-uridine of the ENT for 5 mininhibitor at 37 ◦NBMPRC, in the (used presence at differentiated HepaRG cells were incubated with 24.5 nM [3H]-uridine for 5 min at 37 °C, in the 100or absencenM or 100 of sodiumµM). Uridine and in accumulation the presence or was absence next ofdetermined the ENT inhibitor by scintillat NBMPRion counting. (used at 100Data nM are or presence or absence of sodium and in the presence or absence of the ENT inhibitor NBMPR (used at expressed100 µM). Uridineas pmol accumulation uridine/mg wasprotein next and determined are the means by scintillation ± S.E.M. counting. of values Data from are at expressed least four as 100 nM or 100 µM). Uridine accumulation was next determined by scintillation counting. Data are independentpmol uridine/mg HepaRG protein cell and cultures. are the meansActivities± S.E.M. of nucleoside of values fromtransporters, at least four defined independent by equations HepaRG expressed as pmol uridine/mg protein and are the means ± S.E.M. of values from at least four describedcell cultures. in Materials Activities and of Methods, nucleoside are transporters, indicated by defined double byarrows equations on the described graphs. in Materials and independent HepaRG cell cultures. Activities of nucleoside transporters, defined by equations Methods, are indicated by double arrows on the graphs. Activitiesdescribed ofin MaterialsENT1, ENT2 and Methods,and CNTs are in in primardicated yby cultured double arrows human on hepatocytes the graphs. and HepaRG cells were Activitiesnext calculated of ENT1, using ENT2 Equations and CNTs (2)–(4), in primary and compared cultured humanwith each hepatocytes other (Figure and HepaRG 6). Primary cells humanwereActivities next hepatocytes calculated of ENT1, exhibited using ENT2 Equations the and highest CNTs (2)–(4), CNTin primar activity andy compared cultured, mainly human withrelated each hepatocytes to CNT1 other (Figureas andindicated HepaRG6). Primary above, cells andhumanwere also next hepatocytesthe calculated highest ENT2 exhibited using activity. Equations the highestProliferating (2)–(4), CNT an activity,HepaRGd compared mainly cells displayedwith related each to the other CNT1 highest (Figure as indicated ENT1 6). activity.Primary above, Differentiatedandhuman also hepatocytes the highest HepaRG exhibited ENT2 cells activity. theexhibited highest Proliferating anCNT ENT1 activity HepaRG activity, mainly cells similar displayedrelated to to that CNT1 the highestof as primary indicated ENT1 human activity. above, hepatocytes,Differentiatedand also the whereas highest HepaRG ENT2they cells displayed activity. exhibited Proliferating lower an ENT1 ENT2 activity actiHepaRGvity similar and cells barely to displayed that detectable, of primary the highest if human any, ENT1 CNT hepatocytes, activityactivity. (FigurewhereasDifferentiated 6). they displayed HepaRG lowercells ENT2exhibited activity an andENT1 barely activity detectable, similar if any, to CNTthat activityof primary (Figure human6). hepatocytes, whereas they displayed lower ENT2 activity and barely detectable, if any, CNT activity (Figure 6).

Figure 6. Comparison of CNT (A), ENT1 (B) and ENT2 (C) activities in primary cultured human Figure 6. Comparison of CNT (A), ENT1 (B) and ENT2 (C) activities in primary cultured human hepatocytes and proliferating and differentiated HepaRG cells. CNT, ENT1 and ENT2 activities based hepatocytes and proliferating and differentiated HepaRG cells. CNT, ENT1 and ENT2 activities based on uridine uptake were calculated from values reported in Figure5, using equations described in the onFigure uridine 6. uptakeComparison were calculatedof CNT (A from), ENT1 values (B) repo andrted ENT2 in Figure(C) activities 5, using in equations primary describedcultured humanin the Material and Methods. Data are expressed as pmol uridine/mg protein and are the means ± S.E.M. of Materialhepatocytes and Methods.and proliferating Data are and expressed differentiated as pmol Hepa uridine/mgRG cells. protein CNT, ENT1 and areand the ENT2 means activities ± S.E.M. based of values from four independent human hepatocyte populations and at least four independent HepaRG on uridine uptake were calculated from values reported in Figure 5, using equations described in the valuescell cultures. from four *, pindependent< 0.05 when hu comparedman hepatocyte to primary populations cultured and human at least hepatocytes; four independent #, p < 0.05HepaRG when Material and Methods. Data are expressed as pmol uridine/mg protein and are the means ± S.E.M. of cellcompared cultures. to *, proliferating p < 0.05 when HepaRG compared cells. to primary cultured human hepatocytes; #, p < 0.05 when comparedvalues from to proliferatingfour independent HepaRG human cells. hepatocyte populations and at least four independent HepaRG cell cultures. *, p < 0.05 when compared to primary cultured human hepatocytes; #, p < 0.05 when

compared to proliferating HepaRG cells.

Pharmaceutics 2018, 10, 246 10 of 18 Pharmaceutics 2018, 10, x FOR PEER REVIEW 10 of 18

When considering total nucleoside transporter activity, i.e., the sum of CNT, ENT1ENT1 and ENT2ENT2 activity-based uridineuridine uptake,uptake, ENT1ENT1 activity accountedaccounted forfor more thanthan 90%90% of uridine uptake for both proliferatingproliferating and differentiated HepaRGHepaRG cells (Figure7 7).). ItIt waswas alsoalso thethe mainmain contributorcontributor toto uridineuridine uptake inin primary primary human human hepatocytes, hepatocytes, but but in ain less a markedless marked manner manner (it represented (it represented 72% of 72% total of uridine total uptake).uridine uptake). The relative The contributionrelative contribution of CNT andof CNT ENT2 and activities ENT2 toactivities uridine uptaketo uridine were uptake substantial were (aroundsubstantial 14% (around for each 14% of them)for each only of inthem) primary only culturedin primary human cultured hepatocytes human hepatocytes (Figure7). (Figure 7).

Figure 7. GraphicalGraphical representation representation of of the the relative relative contri contributionbution of of CNTs, CNTs, ENT1 ENT1 an andd ENT2 ENT2 activities activities to tototal total uridine uridine uptake uptake in in proliferating proliferating (A (A) )and and differentiated differentiated ( (BB)) HepaRG HepaRG cells cells and primary humanhuman hepatocytes (C).). PercentagesPercentages indicatedindicated onon graphsgraphs correspondcorrespond toto thethe percentagespercentages ofof contribution.contribution.

3.3. Regulation of Nucleoside Transporter ExpressionExpression byby ProteinProtein KinaseKinase CC (PKC)(PKC) ActivationActivation PKC activation regulates expression of variousvarious sinusoidalsinusoidal and canalicularcanalicular drug transporters in human hepatic cells [[33].33]. To determine whether it may also impact impact nucleoside nucleoside transporter transporter expression, expression, wewe exposedexposed HepaRGHepaRG cellscells to to the the PKCs-activating PKCs-activating agent agent PMA PMA for for various various lengths lengths of of times times (from (from 2 h 2 toh 48to h)48 andh) and analyzed analyzed CNT1, CNT1, ENT1 ENT1 and ENT2 and mRNAENT2 mRNA expression. expression. As shown As inshown Figure in8A–C, Figures treatment 8A-C, bytreatment PMA for by 6 hPMA and for 24 h6 repressedh and 24 CNT1h repressed and ENT2 CNT1 mRNA and ENT2 levels, mRNA and concomitantly levels, and concomitantly induced those ofinduced ENT1; those a shorter of ENT1; exposure a shorter time (2exposure h) also down-regulatedtime (2 h) also down-regulated ENT2, but did notENT2, impact but CNT1did not or impact ENT1 mRNACNT1 or expression, ENT1 mRNA whereas expression, a longer whereas exposure a longer time exposure (48 h) repressed time (48 CNT1,h) repressed without CNT1, significantly without alteringsignificantly expression altering of expression ENT1 or ENT2.of ENT1 Treatment or ENT2. Treatment of primary of human primary hepatocytes human hepatocytes by PMA by for PMA 24 h alsofor 24 reduced h also CNT1 reduced mRNA CNT1 expression, mRNA andexpression, induced and that induced of ENT1 (Figurethat of 8ENT1D); ENT2 (Figure expression 8D); ENT2 was concomitantlyexpression was slightly concomitantly decreased, slightly without decreased, reaching without statistical reaching significance. statistical Treatment significance. of HepaRG Treatment cells byof HepaRG PMA for cells 24 h by was PMA next for shown 24 h was to increasenext shown ENT1 to increase activity ENT1 by a 2.7-fold activity factorby a 2.7-fold (Figure factor8E). Finally,(Figure CNT18E). Finally, mRNA CNT1 repression mRNA by repression PMA was by found PMA towas be found prevented to be byprevented co-treatment by co-treatment with the pan-PKC with the inhibitorpan-PKC GFinhibitor 109203X GF [109203X34], but [34], not withbut not the with PKC th inhibitore PKC inhibitor Gö 6976 Gö (Figure 6976 (Figure8F), that 8F), targets that targets only conventionalonly conventional PKCs PKCs [35]. By[35]. contrast, By contrast, both GFboth 109203X GF 109203X and Gö and 6976 Gö inhibited 6976 inhibited PMA-mediated PMA-mediated ENT1 mRNAENT1 mRNA up-regulation up-regulation (Figure (Figure8G). 8G).

Pharmaceutics 2018, 10, 246 11 of 18 Pharmaceutics 2018, 10, x FOR PEER REVIEW 11 of 18

Figure 8. Effects of the protein kinase C (PKC)-activating agent phorbol myristate acetate (PMA) on Figure 8. Effects of the protein kinase C (PKC)-activating agent phorbol myristate acetate (PMA) on transportertransporter nucleoside nucleoside expression expression in in differentiated differentiated HepaRG HepaRG cells.cells. ((A–C) Differentiated HepaRG HepaRG cells cells werewere exposed exposed to 100 to 100 nM PMAnM PMA for variousfor various lengths lengths of time of time (0 to (0 48 to h). 48 Expression h). Expression of (A of) CNT1,(A) CNT1, (B) ENT1 (B) andENT1 (C) ENT2 and mRNAs(C) ENT2 was mRNAs next analyzed was next by analyzed RT-qPCR. by Data RT-qPC are expressedR. Data are as expressed fold change as comparatively fold change to untreatedcomparatively cells andto untreated are the means cells and± S.E.M. are the of means values ± S.E.M. from four of values independent from four assays. independent *, p < 0.05 assays. when compared*, p < 0.05 to cellswhen not compared exposed to to cells PMA. not ( Dexposed) Primary to PMA. human (D hepatocytes) Primary human were eitherhepatocytes untreated were (control) either or exposeduntreated to (control) 100 nM or PMA. exposed CNT1, to 100 ENT1 nM andPMA. ENT2 CNT1, expression ENT1 and was ENT2 next expression analyzed was by RT-qPCR.next analyzed Data areby expressed RT-qPCR. as Data fold are change expressed comparatively as fold change to untreated comparatively cells andto untreated are the means cells and± areS.E.M. the meansof values ± fromS.E.M. at least of threevalues independent from at least hepatocyte three independent populations. hepatocyte *, p < 0.05populations. when compared *, p < 0.05 to when cells notcompared exposed to PMA.to cells (E not) HepaRG exposed cells to PMA. were (E either) HepaRG untreated cells were or exposed either untreated to 100 nM or PMAexposed for to 24 100 h. ENT1nM PMA activity for was24 next h. ENT1 determined activity as was indicated next dete inrmined Materials as indicated and Methods. in Materials Data are and expressed Methods. asData pmol are uridine/mg expressed proteinas pmol and uridine/mg are the means protein± S.E.M. and ar ofe values the means from ±three S.E.M. independent of values from assays. three *, independentp < 0.05 when assays. compared *, p to control< 0.05 when cells. compared (F,G) HepaRG to control cells cells. were ( eitherF,G) HepaRG untreated cells or were exposed either to untreated 100 nM PMA or exposed for 24 hto in 100 the absencenM PMA or presence for 24 h ofin 2theµM absence GF 109203X or presence or 5 µ ofM 2 Gö µM 6976. GF 109203X (F) CNT1 or and5 µM (G Gö) ENT16976. ( mRNAF) CNT1 expression and (G) wasENT1 next determinedmRNA expression by RT-qPCR. was next Data determined are expressed by asRT-qPCR. fold change Data comparatively are expressed toas untreated fold change cells andcomparatively are the means to± untreatedS.E.M. of cells values and fromare the three means independent ± S.E.M. of values assays. from *, p three< 0.05 independent when compared assays. to control*, p < cells 0.05 notwhen exposed compared to PKC to control inhibitors. cells not exposed to PKC inhibitors.

Pharmaceutics 2018, 10, 246 12 of 18

3.4.Pharmaceutics Regulation 2018 of, Nucleoside10, x FOR PEER Transporter REVIEW Expression by Inflammatory and Growth Factors 12 of 18

3.4.Expression Regulation of Nucleoside human hepatic Transporter drug Expression transporters by Inflammatory is well known and toGrowth be regulated Factors by inflammatory factors, including the inflammatory cytokines IL6, IL1β and TNFα [24], and by growth factors like HGFExpression [36]. To of determine human hepatic whether drug these transporters factors is may well also known impact to be the regulated expression by inflammatory of nucleoside factors, including the inflammatory cytokines IL6, IL1β and TNFα [24], and by growth factors like transporters in human hepatic cells, HepaRG cells were exposed to the inflammatory cytokines HGF [36]. To determine whether these factors may also impact the expression of nucleoside IL1β, IL6 or TNFα, to the reference inflammatory agent LPS or to HGF for 24 h. ENT1, ENT2 transporters in human hepatic cells, HepaRG cells were exposed to the inflammatory cytokines IL1β, and CNT1 mRNA expression was next determined by RT-qPCR (Figure9A–C). Inflammatory IL6 or TNFα, to the reference inflammatory agent LPS or to HGF for 24 h. ENT1, ENT2 and CNT1 cytokines, LPS and HGF decreased expression of CNT1 (Figure9A) and ENT2 (Figure9C). By contrast, mRNA expression was next determined by RT-qPCR (Figures 9A–C). Inflammatory cytokines, LPS β α ENT1and HGF mRNA decreased levels wereexpression not impaired of CNT1 by(Figure IL1 9A), IL6, and TNF ENT2and (Figure LPS, 9C). whereas By contrast, they wereENT1 markedlymRNA inducedlevels were by HGFnot impaired (by a 6.0-fold by IL1β, factor)IL6, TNF (Figureα and LPS,9B). whereas HGF treatment they were was markedly next induced demonstrated by HGF to concomitantly(by a 6.0-fold induce factor) ENT1(Figure activity 9B). HGF (Figure treatment9D). Finally, was next the demonstrated Janus kinase (JAK)to concomitantly inhibitor ruxolitinib, induce whichENT1 has activity previously (Figure been9D). Finally, shown the to reverseJanus kinase IL6-mediated (JAK) inhibitor suppression ruxolitinib, of which the hepatic has previously sinusoidal drugbeen transporters shown to reverse sodium-taurocholate IL6-mediated suppression cotransporting of the polypeptidehepatic sinusoidal (NTCP/ drugSLC10A1 transporters), OATP1B1 sodium- and OCT1taurocholate [37], also cotransporting hindered IL6-mediated polypeptide down-regulation (NTCP/SLC10A1 of), OATP1B1 CNT1 (Figure and OCT19E) and [37], ENT2 also (Figurehindered9F). ByIL6-mediated contrast, it failed down-regulation to block IL1 βof-mediated CNT1 (Figure suppression 9E) and ENT2 of CNT1 (Figure (Figure 9F).9 E)By andcontrast, ENT2 it (Figurefailed to9F) mRNAblock expression.IL1β-mediated suppression of CNT1 (Figure 9E) and ENT2 (Figure 9F) mRNA expression.

Figure 9. Cont.

Pharmaceutics 2018, 10, 246 13 of 18 Pharmaceutics 2018, 10, x FOR PEER REVIEW 13 of 18

Figure 9. Effects of pro-inflammatory and growth factors on nucleoside transporter expression in Figure 9. Effects of pro-inflammatory and growth factors on nucleoside transporter expression in differentiateddifferentiated HepaRG HepaRG cells. cells. ( A(A––CC)) Differentiated Differentiated HepaRG cells cells were were either either untreated untreated (control) (control) or or exposedexposed to 10to ng/mL10 ng/mL IL6, IL6, 1 ng/mL1 ng/mL IL1 IL1ββ,, 1010 ng/mLng/mL TNF TNFαα, ,10 10 ng/mL ng/mL LPS LPS or or20 20ng/mL ng/mL HGF HGF for 24 for h. 24 h. ExpressionExpression of (ofA )(A CNT1,) CNT1, (B ()B ENT1) ENT1 and and ( C(C)) ENT2ENT2 mRNAsmRNAs was was next next analyzed analyzed by by RT-qPCR. RT-qPCR. Data Data are are expressedexpressed as foldas fold change change comparatively comparatively to to untreated untreated controlcontrol cells and are are the the me meansans ± ±S.E.M.S.E.M. of values of values fromfrom at leastat least three three independent independent assays.assays. *, *, p p< 0.05< 0.05 when when compared compared to control to control cells. ( cells.D) HepaRG (D) HepaRG cells cellswere were either either untreated untreated (control) (control) or orexposed exposed to to20 20ng/mL ng/mL HGF HGF for for24 24h. h.ENT1 ENT1 activity activity was was next next determineddetermined as indicatedas indicated in in Materials Materials and and Methods. Methods. DataData are expressed as as pmol pmol uridine/mg uridine/mg protein protein andand are are the the means means± S.E.M.± S.E.M. of of values valuesfrom from threethree independentindependent assays. assays. *, *,p p< <0.05 0.05 when when compared compared to to controlcontrol cells. cells. (E,F ()E HepaRG,F) HepaRG cells cells were were either either untreated untreated (control), (control), exposed exposed to 5 toµM 5 ruxolitinib,µM ruxolitinib, 10 ng/mL 10 IL6ng/mL or 1 ng/mL IL6 or 1 IL1 ng/mLβ or IL1 co-exposedβ or co-exposed to IL6/ruxolitinib to IL6/ruxolitinib or or IL1 IL1β/ruxolitinibβ/ruxolitinib for 24 24 h. h. (E ()E CNT1) CNT1 and and (F) ENT2(F) ENT2 mRNA mRNA expression expression was was next next determined determined by RT-qPCR. RT-qPCR. Data Data are are expressed expressed as asfold fold change change comparativelycomparatively to untreatedto untreated control control cells cells and and are are the the meansmeans ± S.E.M.S.E.M. of of values values from from three three independent independent assays. *, p < 0.05; NS, not significant. assays. *, p < 0.05; NS, not significant.

4. Discussion4. Discussion In theIn the present present study, study, the the human human hepatoma hepatoma HepaRGHepaRG cell cell line line was was characterized characterized with with respect respect to to expression,expression, activity activity and and regulation regulation of of nucleoside nucleoside transporters.transporters. HepaRG HepaRG cells cells cultured cultured in the in the presence presence of DMSO were found to exhibit a nucleoside transporter mRNA profile similar to that found in of DMSO were found to exhibit a nucleoside transporter mRNA profile similar to that found in freshly freshly isolated human hepatocytes or primary human hepatocytes, i.e., they express CNT1, ENT1 isolated human hepatocytes or primary human hepatocytes, i.e., they express CNT1, ENT1 and ENT2, and ENT2, but not CNT2 and CNT3. This likely confirms the highly differentiated status of DMSO- but not CNT2 and CNT3. This likely confirms the highly differentiated status of DMSO-exposed exposed HepaRG cells, which display expression of main hepatic detoxifying proteins [38]. By HepaRGcontrast, cells, CNT1, which unlike display ENT1 expression and ENT2, of main was hepaticnot expressed detoxifying at the proteins mRNA [level38]. Byin contrast,proliferating CNT1, unlikeHepaRG ENT1 cells, and known ENT2, to was exhibit not expresseda low level atof hepatic the mRNA differentiation level in proliferating [1]. Such data HepaRG suggest that cells, CNT1 known to exhibitcan be aconsidered low level as of a hepatic differentiation differentiation marker [for1]. human Such data hepatic suggest cells, thatwhich CNT1 fully can agrees be consideredwith its as apreviously differentiation published marker expression for human profile, hepatic i.e., CNT1 cells, whichexpression fully is agrees restricted with to its the previously liver, kidney published and expressionsmall intestine, profile, whereas, i.e., CNT1 ENT1 expression and ENT2 is restrictedare more widely to the liver,distributed kidney [39]. and The small absence intestine, or very whereas, low ENT1level and of ENT2CNT2 aremRNA more expression widelydistributed in HepaRG [cell39].s and The freshly absence isolated or very hu lowman level hepatocytes of CNT2 agrees mRNA expressionwith previous in HepaRG studies cells which and freshly detected isolated no substant human hepatocytesial transcript agrees expression with previous of this studiesnucleoside which detectedtransporter no substantial in human transcriptliver [40,41] expression and in suspen of thisded nucleoside human hepatocytes transporter [20]. in By human contrast, liver CNT2 [40, 41] andtranscripts in suspended have humanbeen reported hepatocytes in sandwich-culture [20]. By contrast,d human CNT2 hepatocytes transcripts [19], havethus suggesting been reported that in sandwich-culturedthis particular three-dimensional human hepatocytes mode [19 ],of thusculture suggesting for human that hepatocytes this particular may three-dimensional induce CNT2 expression. CNT2 may alternatively be in fact present in the human liver, but only at a level lower mode of culture for human hepatocytes may induce CNT2 expression. CNT2 may alternatively be than those found for ENT1, ENT2 and CNT1 [18]. mRNA expression of CNT3, known to be weakly in fact present in the human liver, but only at a level lower than those found for ENT1, ENT2 and present in the human liver [42], was not detected in both proliferating and differentiated HepaRG CNT1 [18]. mRNA expression of CNT3, known to be weakly present in the human liver [42], was not cells, freshly isolated human hepatocytes and primary cultured hepatocytes; it was similarly present detected in both proliferating and differentiated HepaRG cells, freshly isolated human hepatocytes and primary cultured hepatocytes; it was similarly present at only low or trace level in sandwich-cultured human hepatocytes [19] and suspended human hepatocytes [20]. Pharmaceutics 2018, 10, 246 14 of 18

For proliferating HepaRG cells, expression of ENT1 transcripts was associated with detection of both ENT1 protein at the plasma membrane and ENT1 activity, i.e., sodium-independent and 100 nM NBMPR-inhibitable transport of uridine, which represents nearly the totality of uridine uptake in these cells. ENT1 activity in differentiated HepaRG cells was also the main contributor to total uridine uptake. However, it was much lower than that found in proliferating HepaRG cells. ENT1 protein was, moreover, not obviously detected at the plasma membrane of differentiated HepaRG cells, which may be consistent with a low level of ENT1 expression at this plasma membrane. By contrast, ENT1 was clearly detected in the nuclei of differentiated HepaRG cells, as well as those of proliferating counterparts. Such a nuclear location of ENT1 is rather unusual and has not been previously reported in sandwich-cultured human hepatocytes and in the human liver, which rather mainly express ENT1 at the sinusoidal membrane, although intracellular location in endocytosis vesicles has also been reported [19]. The nuclear labeling in HepaRG cells may consequently be hypothesized to correspond to an artefact due to the non-specific binding of the used anti-ENT1 antibody to an unidentified ENT1-like protein in the nucleus. The fact that immunolocalization with the anti-ENT1 antibody resulted in nuclear labeling of HAP1 and HAP1 ENT14 cells (Figure S2A) likely supports this hypothesis. ENT2 was also found to be located in the nuclei of proliferating and differentiated HepaRG cells, suggesting that ENT2 expression at the plasma membrane is probably very weak, which agrees with the low contribution of ENT2 activity to total uridine transport in HepaRG cells. It is noteworthy that nuclear localization of ENT2 has previously been reported in other cell types [43,44] and may be implicated in nuclear uptake of nucleosides [45]. CNT1 protein was detected in differentiated HepaRG cells, but not in proliferating counterparts, which agrees with the mRNA expression profiles of these cells. CNT1 was however not located at the sinusoidal plasma membrane, but mainly at the pericanalicular or canalicular domain of differentiated HepaRG cells, which may explain why differentiated HepaRG cells failed to exhibit obvious CNT activity, investigated as a sodium-dependent uridine uptake activity in the present study. By contrast, primary human hepatocytes displayed CNT1 activity, i.e., sodium-dependent uptake of uridine inhibited by thymidine, but not by inosine. Such a result is consistent with the expression of CNT1 at the sinusoidal pole of cultured hepatocytes, knowing that a fraction of CNT1 is also located at the canalicular pole [19]. Besides immunolocalization assays, Western-blotting experiments may have to be considered in the future for more precisely quantifying nucleoside transporter protein expression in both HepaRG cells and primary human hepatocytes. This may permit us to search for a putative correlation between protein expression and the activities of nucleoside transporters. PKC activation, well-known to modulate expression of various hepatic drug transporters [23,33], was found to regulate expression of nucleoside transporters in differentiated HepaRG cells and primary cultured human hepatocytes. The effects of PMA however depend on the nature of the nucleoside transporters: The phorbol ester repressed both CNT1 and ENT2 mRNA expression and induced that of ENT1. PMA concomitantly enhanced ENT1 activity in differentiated HepaRG cells, indicating that ENT1 regulation by PKC activation occurs at a functional level. ENT1 up-regulation by PMA was hindered by the pan-PKC inhibitor GF 109203X and by the conventional/classical PKC inhibitor Gö 6976, indicating that ENT1 induction implicates activation of a conventional/classical PKC, most likely the PKC-α isoform, which is the main conventional PKC isoform present in HepaRG cells [23]. By contrast, CNT1 repression by PMA was prevented by GF 109203X, but not by Gö 6976, thus suggesting that it may depend on activation of a novel PKC isoform expressed in HepaRG cells, i.e., PKC-δ, PKC-ε or PKC-η [23]. The implication of atypical PKCs, that lack the tandem repeat of cysteine rich motifs corresponding to the PKC binding domain of phorbol esters like PMA [46], has to be discarded. Inflammatory cytokines such as IL1β, IL6 and TNFα as well as growth factors like HGF constitute other factors known to regulate drug transporter expression in hepatocytes [24,36,47,48], which likely contributes to the overall repression of hepatic detoxification pathways during inflammation [49]. Cytokines have also been demonstrated to regulate expression of nucleoside transporters in certain Pharmaceutics 2018, 10, 246 15 of 18 cell types. CNT1 is thus up-regulated by IL6 and TNFα in rat hepatocytes [50], whereas interferon (IFN) α repressed ENT1 expression and activity in mouse macrophages [51]. ENT1 expression is also suppressed by IL1β in stromal progenitor cells [52], whereas IFNα induced CNT2 expression and activity in the non-transformed human hepatocyte-derived cell line HHL5 [53]. Our data fully confirm the regulation of nucleoside transporter expression by inflammatory cytokines, through demonstrating that IL1β, IL6 and TNFα, as well as the pro-inflammatory factors LPS, decreased ENT2 and CNT1 mRNA expression in differentiated HepaRG cells. The repressing effects of IL6, unlike those of IL1β, were counteracted by the JAK inhibitor ruxolitinib, which is fully consistent with the fact that IL6, but not IL1β, acts through the JAK/Signal transducer and activator of transcription (STAT) signaling pathway on drug transporter expression [37]. Like inflammatory cytokines, HGF reduced CNT1 and ENT2 mRNA expression in HepaRG cells. HGF also markedly increased ENT1 mRNA expression, which, by contrast, was not altered by inflammatory cytokines. HGF concomitantly induced ENT1 activity, indicating that ENT1 up-regulation by HGF was functionally relevant. HGF appears thus as differentially regulating nucleoside transporter in HepaRG cells; it represses CNT1 and ENT2 and induces ENT1. Whether such differential regulations may occur in vivo in response to HGF and what may be their putative physio-pathological consequences in terms of nucleoside transport in human liver would deserve further studies. DMSO-exposed HepaRG cells are now considered as relevant surrogates for human hepatocytes for drug metabolism, transport and toxicity studies [3,38]. However, HepaRG cells remain transformed hepatoma cells, which express at low level certain detoxifying proteins present in human hepatocytes, such as the cytochromes P-450 1A2, 2D6 and 2E1 and the drug transporters OATP1B3 (SLCO1B3) and bile salt export pump (BSEP/ABCB11). The absence of detectable CNT1 activity in differentiated DMSO-exposed HepaRG cells constitutes another difference with primary human hepatocytes. In this context, however, it is noteworthy that ENT1 activity remains the main nucleoside transporter activity contributing to uridine uptake in both differentiated HepaRG cells and primary human hepatocytes. Moreover, ENT1 is considered to be the major contributor to the hepatic uptake of nucleoside analogs such as the antiviral drug ribavirin [20,21]; in this way, ENT1 expression variations may account for differences in response rate in patients receiving ribavirin-based anti-hepatitis C virus therapy [54]. Therefore, such data suggest that the use of differentiated ENT1-expressing HepaRG cells may be relevant for the analysis of cellular pharmacokinetics and metabolism of nucleoside-like drugs, especially of those mainly transported by ENT1. The relevance of HepaRG cells for nucleoside transporters-related studies is also supported by the fact that HepaRG cells exhibit nucleoside transporter regulations in response to various effectors such as PKCs, inflammatory cytokines and the growth factor HGF. In summary, HepaRG cells were found to exhibit mRNA expression of the nucleoside transporters ENT1, ENT2 and CNT1, and to display notable ENT1 activity. Nucleoside transporters, especially ENT1, can consequently be added to the list of drug transporters expressed and functional in human hepatoma HepaRG cells.

Supplementary Materials: The following are available online at http://www.mdpi.com/1999-4923/10/4/246/s1, Figure S1: 18S rRNA expression in proliferating and differentiated HepaRG cells; Figure S2: Nucleoside transporter mRNA expression and uridine uptake in (A) human hepatoma HuH-7 cells and (B) human macrophages; Figure S3: ENT1 expression and uridine uptake in HAP1 and HAP1 ENT14 cells. Author Contributions: Conceptualization, A.M. (Abdullah Mayati), A.M. (Amélie Moreau), C.D., Y.P. and O.F.; methodology, O.F.; formal analysis, A.M. (Abdullah Mayati), E.J., O.F.; investigation, A.M. (Abdullah Mayati), E.J., M.F.-J.; writing—original draft preparation, O.F.; writing—review and editing, A.M. (Abdullah Mayati), A.M. (Amélie Moreau), Y.P. and O.F; supervision, O.F.; project administration, O.F. Funding: This research received no external funding. Acknowledgments: The authors are grateful to Faten Koraïchi-Auriol for the critical reading of the manuscript. Conflicts of Interest: The authors declare no conflict of interest. Pharmaceutics 2018, 10, 246 16 of 18

References

1. Gripon, P.; Rumin, S.; Urban, S.; Le Seyec, J.; Glaise, D.; Cannie, I.; Guyomard, C.; Lucas, J.; Trepo, C.; Guguen-Guillouzo, C. Infection of a human hepatoma cell line by hepatitis B virus. Proc. Natl. Acad. Sci. USA 2002, 99, 15655–15660. [CrossRef][PubMed] 2. Antherieu, S.; Chesne, C.; Li, R.; Guguen-Guillouzo, C.; Guillouzo, A. Optimization of the HepaRG cell model for drug metabolism and toxicity studies. Toxicol. In Vitro 2012, 26, 1278–1285. [CrossRef][PubMed] 3. Andersson, T.B.; Kanebratt, K.P.; Kenna, J.G. The HepaRG cell line: A unique in vitro tool for understanding drug metabolism and toxicology in human. Expert. Opin. Drug Metab. Toxicol. 2012, 8, 909–920. [CrossRef] [PubMed] 4. Aninat, C.; Piton, A.; Glaise, D.; Le Charpentier, T.; Langouet, S.; Morel, F.; Guguen-Guillouzo, C.; Guillouzo, A. Expression of cytochromes P450, conjugating enzymes and nuclear receptors in human hepatoma HepaRG cells. Drug Metab. Dispos. 2006, 34, 75–83. [CrossRef][PubMed] 5. Le Vee, M.; Jigorel, E.; Glaise, D.; Gripon, P.; Guguen-Guillouzo, C.; Fardel, O. Functional expression of sinusoidal and canalicular hepatic drug transporters in the differentiated human hepatoma HepaRG cell line. Eur. J. Pharm. Sci. 2006, 28, 109–117. [CrossRef][PubMed] 6. Le Vee, M.; Noel, G.; Jouan, E.; Stieger, B.; Fardel, O. Polarized expression of drug transporters in differentiated human hepatoma HepaRG cells. Toxicol. In Vitro 2013, 27, 1979–1986. [CrossRef][PubMed] 7. Le Vee, M.; Jouan, E.; Noel, G.; Stieger, B.; Fardel, O. Polarized location of SLC and ABC drug transporters in monolayer-cultured human hepatocytes. Toxicol. In Vitro 2015, 29, 938–946. [CrossRef][PubMed] 8. Molina-Arcas, M.; Casado, F.J.; Pastor-Anglada, M. Nucleoside transporter proteins. Curr. Vasc. Pharmacol. 2009, 7, 426–434. [CrossRef][PubMed] 9. Young, J.D.; Yao, S.Y.; Baldwin, J.M.; Cass, C.E.; Baldwin, S.A. The human concentrative and equilibrative nucleoside transporter families, SLC28 and SLC29. Mol. Aspects Med. 2013, 34, 529–547. [CrossRef][PubMed] 10. Gray, J.H.; Owen, R.P.; Giacomini, K.M. The concentrative nucleoside transporter family, SLC28. Pflugers Arch. 2004, 447, 728–734. [CrossRef][PubMed] 11. Baldwin, S.A.; Beal, P.R.; Yao, S.Y.; King, A.E.; Cass, C.E.; Young, J.D. The equilibrative nucleoside transporter family, SLC29. Pflugers Arch. 2004, 447, 735–743. [PubMed] 12. Baldwin, S.A.; Yao, S.Y.; Hyde, R.J.; Ng, A.M.; Foppolo, S.; Barnes, K.; Ritzel, M.W.; Cass, C.E.; Young, J.D. Functional characterization of novel human and mouse equilibrative nucleoside transporters (hENT3 and mENT3) located in intracellular membranes. J. Biol. Chem. 2005, 280, 15880–15887. [CrossRef][PubMed] 13. Engel, K.; Wang, J. Interaction of organic cations with a newly identified plasma membrane monoamine transporter. Mol. Pharmacol. 2005, 68, 1397–1407. [CrossRef][PubMed] 14. Zhou, M.; Duan, H.; Engel, K.; Xia, L.; Wang, J. Adenosine transport by plasma membrane monoamine transporter: Reinvestigation and comparison with organic cations. Drug Metab. Dispos. 2010, 38, 1798–1805. [CrossRef][PubMed] 15. Pastor-Anglada, M.; Cano-Soldado, P.; Errasti-Murugarren, E.; Casado, F.J. SLC28 genes and concentrative nucleoside transporter (CNT) proteins. Xenobiotica 2008, 38, 972–994. [CrossRef][PubMed] 16. Kong, W.; Engel, K.; Wang, J. Mammalian nucleoside transporters. Curr. Drug Metab. 2004, 5, 63–84. [CrossRef][PubMed] 17. Pastor-Anglada, M.; Perez-Torras, S. Nucleoside transporter proteins as biomarkers of drug responsiveness and drug targets. Front. Pharmacol. 2015, 6, 13. [CrossRef][PubMed] 18. Govindarajan, R.; Bakken, A.H.; Hudkins, K.L.; Lai, Y.; Casado, F.J.; Pastor-Anglada, M.; Tse, C.M.; Hayashi, J.; Unadkat, J.D. In situ hybridization and immunolocalization of concentrative and equilibrative nucleoside transporters in the human intestine, liver, kidneys, and placenta. Am. J. Physiol. Regul. Integr. Comp. Physiol. 2007, 293, R1809–R1822. [CrossRef][PubMed] 19. Govindarajan, R.; Endres, C.J.; Whittington, D.; LeCluyse, E.; Pastor-Anglada, M.; Tse, C.M.; Unadkat, J.D. Expression and hepatobiliary transport characteristics of the concentrative and equilibrative nucleoside transporters in sandwich-cultured human hepatocytes. Am. J. Physiol. Gastrointest. Liver Physiol. 2008, 295, G570–G580. [CrossRef][PubMed] 20. Fukuchi, Y.; Furihata, T.; Hashizume, M.; Iikura, M.; Chiba, K. Characterization of ribavirin uptake systems in human hepatocytes. J. Hepatol. 2010, 52, 486–492. [CrossRef][PubMed] Pharmaceutics 2018, 10, 246 17 of 18

21. Choi, M.K.; Kim, M.H.; Maeng, H.J.; Song, I.S. Contribution of CNT1 and ENT1 to ribavirin uptake in human hepatocytes. Arch. Pharm. Res. 2015, 38, 904–913. [CrossRef][PubMed] 22. Shimada, T.; Nakanishi, T.; Tajima, H.; Yamazaki, M.; Yokono, R.; Takabayashi, M.; Sawamoto, K.; Miyamoto, K.; Kitagawa, H.; Ohta, T.; et al. Saturable Hepatic Extraction of Gemcitabine Involves Biphasic Uptake Mediated by Nucleoside Transporters Equilibrative Nucleoside Transporter 1 and 2. J. Pharm. Sci. 2015, 104, 3162–3169. [CrossRef][PubMed] 23. Mayati, A.; Le Vee, M.; Moreau, A.; Jouan, E.; Bucher, S.; Stieger, B.; Denizot, C.; Parmentier, Y.; Fardel, O. Protein kinase C-dependent regulation of human hepatic drug transporter expression. Biochem. Pharmacol. 2015, 98, 703–717. [CrossRef][PubMed] 24. Fardel, O.; Le Vee, M. Regulation of human hepatic drug transporter expression by pro-inflammatory cytokines. Expert Opin. Drug Metab. Toxicol. 2009, 5, 1469–1481. [CrossRef][PubMed] 25. Jouan, E.; Le Vee, M.; Denizot, C.; Parmentier, Y.; Fardel, O. Drug Transporter Expression and Activity in Human Hepatoma HuH-7 Cells. Pharmaceutics 2016, 9, 3. [CrossRef][PubMed] 26. Van Grevenynghe, J.; Rion, S.; Le Ferrec, E.; Le Vee, M.; Amiot, L.; Fauchet, R.; Fardel, O. Polycyclic aromatic hydrocarbons inhibit differentiation of human monocytes into macrophages. J. Immunol. 2003, 170, 2374–2381. [CrossRef][PubMed] 27. Bustin, S.A.; Benes, V.; Garson, J.A.; Hellemans, J.; Huggett, J.; Kubista, M.; Mueller, R.; Nolan, T.; Pfaffl, M.W.; Shipley, G.L.; et al. The MIQE guidelines: Minimum information for publication of quantitative real-time PCR experiments. Clin. Chem. 2009, 55, 611–622. [CrossRef][PubMed] 28. Le Vee, M.; Gripon, P.; Stieger, B.; Fardel, O. Down-regulation of organic anion transporter expression in human hepatocytes exposed to the proinflammatory cytokine interleukin 1beta. Drug Metab. Dispos. 2008, 36, 217–222. [CrossRef][PubMed] 29. Mayati, A.; Moreau, A.; Denizot, C.; Stieger, B.; Parmentier, Y.; Fardel, O. beta2-adrenergic -mediated in vitro regulation of human hepatic drug transporter expression by epinephrine. Eur. J. Pharm. Sci. 2017, 106, 302–312. [CrossRef][PubMed] 30. Klein, D.M.; Evans, K.K.; Hardwick, R.N.; Dantzler, W.H.; Wright, S.H.; Cherrington, N.J. Basolateral uptake of nucleosides by Sertoli cells is mediated primarily by equilibrative nucleoside transporter 1. J. Pharmacol. Exp. Ther. 2013, 346, 121–129. [CrossRef][PubMed] 31. Furihata, T.; Fukuchi, Y.; Iikura, M.; Hashizume, M.; Miyajima, A.; Nagai, M.; Chiba, K. Striking species difference in the contribution of concentrative nucleoside transporter 2 to nucleoside uptake between mouse and rat hepatocytes. Antimicrob. Agents Chemother. 2010, 54, 3035–3038. [CrossRef][PubMed] 32. Bachour-El Azzi, P.; Sharanek, A.; Burban, A.; Li, R.; Guevel, R.L.; Abdel-Razzak, Z.; Stieger, B.; Guguen-Guillouzo, C.; Guillouzo, A. Comparative Localization and Functional Activity of the Main Hepatobiliary Transporters in HepaRG Cells and Primary Human Hepatocytes. Toxicol. Sci. 2015, 145, 157–168. [CrossRef][PubMed] 33. Mayati, A.; Moreau, A.; Le Vee, M.; Stieger, B.; Denizot, C.; Parmentier, Y.; Fardel, O. Protein Kinases C-Mediated Regulations of Drug Transporter Activity, Localization and Expression. Int. J. Mol. Sci. 2017, 18, 764. [CrossRef][PubMed] 34. Toullec, D.; Pianetti, P.; Coste, H.; Bellevergue, P.; Grand-Perret, T.; Ajakane, M.; Baudet, V.; Boissin, P.; Boursier, E.; Loriolle, F.; et al. The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of protein kinase C. J. Biol. Chem. 1991, 266, 15771–15781. [PubMed] 35. Martiny-Baron, G.; Kazanietz, M.G.; Mischak, H.; Blumberg, P.M.; Kochs, G.; Hug, H.; Marme, D.; Schachtele, C. Selective inhibition of protein kinase C isozymes by the indolocarbazole Go 6976. J. Biol. Chem. 1993, 268, 9194–9197. [PubMed] 36. Le Vee, M.; Lecureur, V.; Moreau, A.; Stieger, B.; Fardel, O. Differential regulation of drug transporter expression by hepatocyte growth factor in primary human hepatocytes. Drug Metab. Dispos. 2009, 37, 2228–2235. [CrossRef][PubMed] 37. Febvre-James, M.; Bruyere, A.; Le Vee, M.; Fardel, O. The JAK1/2 Inhibitor Ruxolitinib Reverses Interleukin-6-Mediated Suppression of Drug-Detoxifying Proteins in Cultured Human Hepatocytes. Drug Metab. Dispos. 2018, 46, 131–140. [CrossRef][PubMed] 38. Guillouzo, A.; Corlu, A.; Aninat, C.; Glaise, D.; Morel, F.; Guguen-Guillouzo, C. The human hepatoma HepaRG cells: A highly differentiated model for studies of liver metabolism and toxicity of xenobiotics. Chem. Biol. Interact. 2007, 168, 66–73. [CrossRef][PubMed] Pharmaceutics 2018, 10, 246 18 of 18

39. Pennycooke, M.; Chaudary, N.; Shuralyova, I.; Zhang, Y.; Coe, I.R. Differential expression of human nucleoside transporters in normal and tumor tissue. Biochem. Biophys. Res. Commun. 2001, 280, 951–959. [CrossRef][PubMed] 40. Ritzel, M.W.; Yao, S.Y.; Ng, A.M.; Mackey, J.R.; Cass, C.E.; Young, J.D. Molecular cloning, functional expression and chromosomal localization of a cDNA encoding a human Na+/nucleoside (hCNT2) selective for purine nucleosides and uridine. Mol. Membr. Biol. 1998, 15, 203–211. [PubMed] 41. Mele, M.; Ferreira, P.G.; Reverter, F.; DeLuca, D.S.; Monlong, J.; Sammeth, M.; Young, T.R.; Goldmann, J.M.; Pervouchine, D.D.; Sullivan, T.J.; et al. Human genomics. The human transcriptome across tissues and individuals. Science 2015, 348, 660–665. [CrossRef][PubMed] 42. Ritzel, M.W.; Ng, A.M.; Yao, S.Y.; Graham, K.; Loewen, S.K.; Smith, K.M.; Ritzel, R.G.; Mowles, D.A.; Carpenter, P.; Chen, X.Z.; et al. Molecular identification and characterization of novel human and mouse concentrative Na+-nucleoside cotransporter proteins (hCNT3 and mCNT3) broadly selective for purine and pyrimidine nucleosides (system cib). J. Biol. Chem. 2001, 276, 2914–2927. [CrossRef][PubMed] 43. Hansen, J.E.; Tse, C.M.; Chan, G.; Heinze, E.R.; Nishimura, R.N.; Weisbart, R.H. Intranuclear protein transduction through a nucleoside salvage pathway. J. Biol. Chem. 2007, 282, 20790–20793. [CrossRef] [PubMed] 44. Klein, D.M.; Harding, M.C.; Crowther, M.K.; Cherrington, N.J. Localization of nucleoside transporters in rat epididymis. J. Biochem. Mol. Toxicol. 2017, 31, e21911. [CrossRef][PubMed] 45. Grane-Boladeras, N.; Spring, C.M.; Hanna, W.J.; Pastor-Anglada, M.; Coe, I.R. Novel nuclear hENT2 isoforms regulate cell cycle progression via controlling nucleoside transport and nuclear reservoir. Cell. Mol. Life Sci. 2016, 73, 4559–4575. [CrossRef][PubMed] 46. Burns, D.J.; Bell, R.M. Protein kinase C contains two phorbol ester binding domains. J. Biol. Chem. 1991, 266, 18330–18338. [PubMed] 47. Hartmann, G.; Kim, H.; Piquette-Miller, M. Regulation of the hepatic multidrug resistance gene expression by endotoxin and inflammatory cytokines in mice. Int. Immunopharmacol. 2001, 1, 189–199. [CrossRef] 48. Diao, L.; Li, N.; Brayman, T.G.; Hotz, K.J.; Lai, Y. Regulation of MRP2/ABCC2 and BSEP/ABCB11 expression in sandwich cultured human and rat hepatocytes exposed to inflammatory cytokines TNF-α, IL-6, and IL-1β. J. Biol. Chem. 2010, 285, 31185–31192. [CrossRef][PubMed] 49. Aitken, A.E.; Richardson, T.A.; Morgan, E.T. Regulation of drug-metabolizing enzymes and transporters in inflammation. Annu. Rev. Pharmacol. Toxicol. 2006, 46, 123–149. [CrossRef][PubMed] 50. Fernandez-Veledo, S.; Valdes, R.; Wallenius, V.; Casado, F.J.; Pastor-Anglada, M. Up-regulation of the high-affinity pyrimidine-preferring nucleoside transporter concentrative nucleoside transporter 1 by tumor necrosis factor-alpha and interleukin-6 in liver parenchymal cells. J. Hepatol. 2004, 41, 538–544. [CrossRef] [PubMed] 51. Soler, C.; Felipe, A.; Garcia-Manteiga, J.; Serra, M.; Guillen-Gomez, E.; Casado, F.J.; MacLeod, C.; Modolell, M.; Pastor-Anglada, M.; Celada, A. Interferon-gamma regulates nucleoside transport systems in macrophages through signal transduction and activator of transduction factor 1 (STAT1)-dependent and -independent signalling pathways. Biochem. J. 2003, 375, 777–783. [CrossRef][PubMed] 52. Ezura, Y.; Lin, X.; Hatta, A.; Izu, Y.; Noda, M. Interleukin-1beta Suppresses the Transporter Genes Ank and Ent1 Expression in Stromal Progenitor Cells Retaining Mineralization. Calcif. Tissue Int. 2016, 99, 199–208. [CrossRef][PubMed] 53. Pinilla-Macua, I.; Fernandez-Calotti, P.; Perez-Del-Pulgar, S.; Pastor-Anglada, M. Ribavirin uptake into human hepatocyte HHL5 cells is enhanced by interferon-alpha via up-regulation of the human concentrative nucleoside transporter (hCNT2). Mol. Pharm. 2014, 11, 3223–3230. [CrossRef][PubMed] 54. Baloch, K.; Chen, L.; Memon, A.A.; Dexter, L.; Irving, W.; Ilyas, M.; Thomson, B.J. Equilibrative nucleoside transporter 1 expression in primary human hepatocytes is highly variable and determines uptake of ribavirin. Antivir. Chem. Chemother. 2017, 25, 2–10. [CrossRef][PubMed]

© 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).