Glucocorticoid receptor regulates organic cation transporter 1 (OCT1, SLC22A1) expression via HNF4a upregulation in primary human hepatocytes
Alice Rulcova1*, Lucie Krausova1*, Tomas Smutny1, Radim Vrzal2, Zdenek Dvorak2, Ramiro Jover3,4, Petr Pavek1
1Department of Pharmacology and Toxicology, Faculty of Pharmacy in Hradec Kralove, Charles University in Prague, Heyrovskeho 1203, Hradec Kralove, CZ-500 05, Czech Republic
2Department of Cell Biology and Genetics, Faculty of Science, Palacky University in Olomouc, Slechtitelu 11, Olomouc, CZ-783 71, Czech Republic
3Unidad Mixta Hepatología Experimental, Hospital La Fe & University of Valencia (Dep. Biochemistry and Molecular Biology), Av. Campanar, 21, 46009 Valencia, Spain
4CIBERehd, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas, Barcelona, Spain Correspondence: Petr Pavek, e-mail: petr.pavek@faf.cuni.cz
Abstract:
Background: Organic cation transporter 1 (OCT1, SLC22A1) is a membrane transporter that is important for therapeutic effect of the antidiabetic drug metformin. Its liver-specific expression in hepatocytes is strongly controlled by hepatocyte nuclear factor-4a (HNF4a). HNF4a expression and transcriptional activity have been demonstrated to be augmented by glucocorticoid receptor (GR) in human hepatocytes and rodent livers.
Methods: It was examined whether GR activation indirectly induces OCT1 gene expression via HNF4a up-regulation in primary human hepatocytes. We also examined which other transcription factors are involved in OCT1 gene expression and whether they are regulated by dexamethasone using qRT-PCR and gene reporter assays.
Results: We found that dexamethasone significantly up-regulates OCT1 mRNA and protein in normal primary human hepatocytes, but not in hepatocyte-derived tumor cell lines HepG2 and MZ-Hep1. Consistently, we observed that HNF4a is induced by dex- amethasone in primary human hepatocytes, but not in hepatocyte tumor-derived cell lines. Viral transduction of MZ-Hep1 cells with the expression constructs for HNF4a, CCAAT/enhancer binding proteins b (C/EBPb) and peroxisome proliferator-activated recep- tor-g coactivator 1a (PGC1a) demonstrated significant roles of the transcription factors in OCT1 gene regulation. We found that ex- pression of OCT1 mRNA in human livers significantly correlates with C/EBPb and HNF4a mRNAs expression and that C/EBPb co-transfection stimulates OCT1 gene reporter construct in HepG2 cells. Nevertheless, neither C/EBPb nor PGC1a were up- regulated in human hepatocytes by dexamethasone.
Conclusion: We can conclude that GR-induced expression of HNF4a may contribute to indirect OCT1 gene up-regulation by dex- amethasone in primary human hepatocytes, but not in hepatocyte-derived tumor cell lines.
Key words:
OCT1 transporter, glucocorticoid receptor, HNF4a, C/EBPb, gene regulation, hepatocyte
Pharmacological Reports 2013, 65, 13221335 ISSN 1734-1140
Copyright © 2013 by Institute of Pharmacology Polish Academy of Sciences
* A.R. and L.K. contributed equally to these studies.
Introduction
Organic cation transporter 1 (OCT1, SLC22A1) be- longs to the solute carrier family of membrane trans- port proteins (SLC). OCT1 is responsible for the up- take of organic cationic compounds from blood to hepatocytes [16, 36]. OCT1 transports numerous cati- onic drugs into hepatocytes, including metformin, amantadine, several antiviral drugs, platinum deri- vates, and mycotoxins [12]. The activity of OCT1 may determine the antidiabetic effect of metformin, the most widely prescribed drug for the therapy of the type 2 diabetes [2, 26, 31].
OCT1 is expressed only in normal human hepatocytes.
Its hepatocyte-specific expression is controlled mainly by the HNF4a nuclear receptor via two DR-2 sites [23].
HNF4a is a transcription factor that belongs to the steroid/thyroid hormone receptor superfamily. It binds, as a homodimer to cis-regulatory promoter sequences of liver-specific genes [3, 24]. Functionally, HNF4a plays a critical role in development, cell differentiation, xenobiotic detoxification, bile acid synthesis, serum protein production and metabolism [32]. A rare loss- of-function mutation in the HNF4a gene causes a monogenic form of early-onset type 2 diabetes (maturity-onset diabetes of the young, MODY1) [34].
HNF4a belongs, together with other transcription factors such as CCAAT/enhancer binding proteins a and b (C/EBPa and C/EBPb) and hepatocyte nuclear factor 1a (HNF1a) and 3g (HNF3g, FOXA3, fork- head box A3), to the family of so called liver-enriched transcription factors (LETFs). LETFs play a special role in hepatic phenotype and differentiation and in the transcriptional regulation of hepatic drug- metabolizing enzymes and drug transporters [4, 11].
Using an adenovirus-mediated HNF4a-small inter- fering RNA in primary human hepatocytes, it was re- cently found that OCT1 is the drug transporter whose expression is most significantly affected by HNF4a silencing [11]. Thus, we can suppose that stimulation of HNF4a transcriptional activity can primarily influ- ence OCT1 expression and hepatic uptake of cationic substrates into the liver.
Recently, our laboratories and others have reported that glucocorticoids up-regulate HNF4a in human and rodent hepatocytes. We found that dexametha- sone significantly up-regulated the mRNA of HNF4a in primary human hepatocytes. The HNF4a mRNA induction by dexamethasone was totally inhibited by the glucocorticoid receptor antagonist RU486 [30].
HNF4a mRNA up-regulation by glucocorticoids was also described by others in primary human hepato- cytes [7, 17], in primary rat hepatocytes and in rat he- patic organoid cultures [14, 18]. Notably, Onica et al.
did not observe HNF4a protein up-regulation in hu- man hepatocytes. Nevertheless, they discovered that dexamethasone increased HNF4a binding to the HNF4 response element of CYP2A6 gene in primary human hepatocytes [17].
These results prompted us to examine whether glu- cocorticoids may indirectly induce OCT1 gene ex- pression in primary human hepatocytes via glucocor- ticoid receptor-mediated HNF4a up-regulation. In ad- dition, we examined whether additional LETFs may be involved in OCT1 gene regulation.
Materials and Methods
Cells
Human hepatoma cells (Mz-Hep1, HepG2) were cul- tured in Ham’s F-12/Leibovitz L-15 (1/1, v/v) me- dium supplemented with 6% fetal bovine serum and cultured to 70–80% confluence. The HepG2 cell line was purchased from the European Collection of Cell Cultures (Salisbury, UK) and was used within 25 pas- sages from delivery. For transient transfection gene reporter experiments, HepG2 cells were maintained in antibiotic-free Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum (FCS), 1%
sodium pyruvate, and 1% nonessential amino acids (all from Sigma-Aldrich, St. Louis, MO, USA). The final concentration of DMSO in culture media was 0.1% (v/v) in all of the experiments.
HepG2 and MZ-Hep1 cells were treated in char- coal-stripped media. Other chemicals were of the highest quality commercially available. Dexametha- sone base and RU486 were purchased from Sigma- Aldrich (St. Louis, MO, USA).
Adenoviral transduction
Recombinant adenoviral vectors of human HNF4a and C/EBPb-LAP were developed as described earlier [10, 13]. The adenoviral vector for the expression of PGC1a was kindly provided by Dr. P. Puigserver (Johns Hop- kins University School of Medicine, Baltimore, MD,
USA) [35]. The viruses were amplified and then puri- fied using the Vivapure kit (Sartorius AG, Göttingen, Germany). Mz-Hep1 cells were infected with recom- binant adenoviruses for 120 min at a multiplicity of infection (M.O.I.) ranging from 1 to 20 plaque- forming units/cell. Forty-eight hours after transfec- tion, mRNA was isolated as described earlier [13].
DNA constructs
To produce a 1.7 kb OCT1 reporter construct with two DR-2 HNF4a binding sites [23], the promoter sequences -1649 to +102, -1458 to +102 and -430 to +102 were synthesized and inserted into the pGL4.10 vector (Promega) using KpnI and XhoI restriction enzymes.
Construct pOCT1(-1649/+102)-luc contains both DR-2 elements binding HNF4a. pOCT1(-1458/+102) DHNF4aRE-luc construct lacks the first HNF4a re- sponse element (A) critical for OCT1 promoter trans- activation with HNF4a. Construct OCT1(-430/+102)- luc lacks both DR-2 elements [23].
Transient transfection experiments were performed in 48-well plates using Transfection reagent (BioRad, Hercules, CA, USA) as described before [22]. Bec- ause hepatocyte-derived tumor cell lines have low GR activity and expression [6], we cotransfected MZ- Hep1 cells with the pSG5-hGRa expression construct (a gift from Dr. Palvimo, University of Helsinki, Hel- sinki, Finland) before the treatment with dexametha- sone [15]. Expression construct for C/EBPb-LAP has been described in our previous paper [10]. A chimeric luciferase reporter construct, containing three in- tandem copies of the HNF4a response element for human ApoCIII in front of a TK promoter (pGL3-B- 3xApoCIII-TK-luc) and its control reporter vector (pGL3-B-TK-LUC) were kindly provided by Dr. I.
Talianidis (Institute of Molecular Biology and Bio- technology, Crete, Greece). pGRE-luc (PathDetect pGRE-Luc Plasmid) GR responsive reporter construct was purchased from Agilent Technologies.
Primary cultures of human hepatocytes
Hepatocytes were prepared from lobectomy segments that had been resected from adult patients. The tissue acquisition protocol was in accordance with the re- quirements issued by local ethical commissions in Spain and the Czech Republic. We did not use steatic or cirrhotic liver tissue, and all of the donors had negative virology for HIV, HCV, EBP, and CMV. He-
patocytes were isolated and cultured as described pre- viously [9, 21]. The cultures were allowed to stabilize for 48 h prior to the treatments. The standard culture medium developed for primary cultures of human he- patocytes contained 100 nM of the glucocorticoid dexamethasone [9]. This cultivation condition is re- ferred as “+Dex”. In other experiments, DEX was re- moved from the culture media 16 h prior to treat- ments. Treatments were performed using DEX-free culture media. This condition is referred to as “-Dex”
(Figs. 1 and 4). Expression of OCT1, C/EBPb or PGC1a mRNAs were compared with control hepato- cyte sample cultivated in the regular medium with 100 nm Dex (+Dex, first column).
Several batches of long-term primary human hepa- tocytes grown in monolayers (Hep220650, 220633, 220624) were obtained from Biopredic, Rennes, France.
We also used liver samples from 42 different do- nors collected by Unidad de Hepatología Experimen- tal (Hospital La Fe, Valencia, Human Liver Bank, HLaFe-CIBERehd) to investigate the correlation be- tween HNF4a, C/EBPb or PGC1a mRNA levels and OCT1 mRNA levels (Fig. 3).
Quantification of mRNA expression employing qRT-PCR
Total RNA was isolated from cells using an RNeasy Total RNA Kit (Qiagen). One microgram of the total RNA was transcribed by M-MLV Reverse Transcrip- tase (Invitrogen). The resulting diluted cDNA was amplified in a LightCycler Instrument with Fast Start DNA Master SYBR Green I (Roche). PCR conditions specific for each pair of primers (Tab. 1) were opti- mized with respect to the MgCl2 concentration, an- nealing temperature, duration of extension and number of cycles. Whenever possible, primer se- quences were chosen to span exon boundaries. PCR fragment size and purity were evaluated by melting curve analysis. In parallel, the human housekeeping isoform of porphobilinogen deaminase (PBGD) cDNA was analyzed for normalization of mRNA expression. Moreover, in each amplification, we included a reference calibration cDNA made from 18 human livers (Human Liver Bank, HLaFe- CIBERehd). The real-time monitoring of the PCR re- action and the precise quantification of the products in the exponential phase of the amplification were per- formed using LightCycler quantification software ac- cording to the manufacturer’s recommendations. Re-
producibility of the measurements was assessed by conducting triplicate reactions.
Western blotting analysis
Immunodetection of OCT1, HNF4a and PGC1a in primary human hepatocyte lysates or in MZ-Hep1 or HepG2 cells was performed according to the western blotting protocol described previously [30].
Total hepatocyte lysates were prepared in RIPA buffer (150 mM NaCl, 25 mM Tris-HCl, 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS, pH 7.6) containing protease inhibitors (Roche, Praha, Czech Republic). The samples were incubated 30 min on ice, vortexed and centrifuged for 10 min at 10,000 × g at 4°C. Supernatants were transferred into new tubes and diluted to equal concentration in RIPA lysis buffer (100 mM HEPES, 15 mM MgCl2, 100 mM KCl, pH 7.9) including dithiothreitol and protease inhibitors.
After electrophoretic resolution and transfer onto membranes, primary rabbit polyclonal antibodies against OCT1, HNF4a, and PGC1a were used. The rabbit polyclonal antibody against human OCT1 (SLC22A1, ab55916) was obtained from Abcam, Cambridge, UK. Antibodies against HNF4a (rabbit polyclonal; sc-8987, H-171) and PGC-1a (goat poly- clonal; sc-8987, H-171) were purchased from Santa Cruz Biotechnology (Santa Cruz, USA). b-Actin was determined as a control in all western blotting experi- ments using I-19 goat polyclonal antipody (Santa Cruz Biotechnology, USA). Chemiluminescence detection was performed using a horseradish peroxidase-con- jugated secondary antibody and the Amersham ECL kit (GE Healthcare).
Statistical analysis
A one-way ANOVA followed by Bonferroni’s multi- ple comparison post hoc tests or the paired Student’s t-test were used for statistical analyses with GraphPad Prism software (GraphPad Software Inc., San Diego, CA, USA). The Pearson correlation coefficient (r) was calculated to measure the strength of the linear relationship between expression of LETFs and OCT1 mRNAs in human hepatocytes.
Results
The glucocorticoid dexamethasone induces OCT1 expression in primary human
hepatocytes
In first series of experiments, we treated four cultures of primary human hepatocytes with dexamethasone (Dex 100 nM), RU486 (1 mM, a GR antagonist) or their combination in either standard Isom medium with glucocorticoids (+Dex medium) or glucocorti- coid-free medium (-Dex medium). We found that RU486 significantly (p < 0.001, ANOVA with Bon- ferroni’s post hoc test) suppressed OCT1 mRNA ex- pression in all four hepatocyte preparations cultivated in +Dex medium (Fig. 1A, liver-to-liver presentation of the data). In the –Dex medium, Dex treatment con- sistently and significantly (p < 0.001) up-regulated OCT1 mRNA expression, while the induction was suppressed by RU486 (Fig. 1A). RU486 itself had no effect on OCT1 mRNA expression in primary human hepatocytes cultivated in the –Dex medium.
Subsequently, we investigated the effect of dex- amethasone on OCT1 protein expression. Consis- tently with qRT-PCR data, we observed that dex- amethasone slightly up-regulated OCT1 protein levels in three primary human hepatocytes after 48 h of treatment (Fig. 1B).
Dexamethasone does not induce the OCT1 gene expression and transactivation in hepatocyte-derived tumor cell lines
Next, we used the hepatoblastoma HepG2 and the he- patocarcinoma MZ-Hep1 cell lines and treated them with Dex (100 nM), RU486 (1 mM) or their combina- tion. We found that Dex does not induce OCT1
Tab. 1. Primer sequences for qRT-PCR analyses of target gene ex- pression
OCT1 Forward Reverse
5’-CGC CGA GAA CCT TGG GAG AAA-3’
5’-ACG ACA TCG CCG CAA AAC ATC-3’
HNF4a Forward Reverse
5’-GCC TAC CTC AAA GCC ATC AT-3’
5’-GAC CCT CCC AGC AGC ATC TC-3’
PGC1a Forward Reverse
5’-AAT GTG TCT CCT TCT TGT TCT T-3’
5’-GGT GTC TGT AGT GGC TTG TTG A-3’
C/EBPb Forward Reverse
5’-CTC GCA GGT CAA GAG CAA G-3’
5’-CTA GCA GTG GCC GGA GGA GGC GAC C-3’
mRNA even after co-transfection of the cell lines with the GR expression construct and treatment with Dex (Fig. 2A). In agreement, Dex did not activate the gene reporter luciferase construct within the 5’-flanking se- quence (-1649/+102) of the OCT1 gene with both DR-2 HNF4a response elements, even after cotransfection of cells with a GR expression construct in HepG2 cells
(Fig. 2B). Control pGRE-luc GR responsive reporter construct was significantly activated by dexametha- sone (about 45-fold activation with 100 nM Dex) un- der the same experimental condition in HepG2 cells.
Finally, we analyzed OCT1 protein expression in MZ-Hep1 cells after treatment with Dex using west- ern blotting. We observe low expression of OCT1
Fig. 1. Effects of dexamethasone on OCT1 mRNA and protein expression in primary human hepatocytes. (A) Four primary human hepatocyte cultures LH155, 157, 164 and 167 were maintained for 16 h in standard Isom medium (+Dex medium) or dexamethasone-free medium (-Dex medium) and subsequently treated for 24 h with RU486 (1 mM), dexamethasone (Dex; 100 nM), the combination of dexamethasone and RU486 or vehicle control (0.1% DMSO). Total RNA was isolated and mRNA levels were analyzed using qRT-PCR. The effects of compounds on OCT1 gene mRNA levels are presented as fold mRNA expression relative to control vehicle-treated cells cultivated in +Dex medium (set to a value of 1). Data are presented as the means of three qRT-PCR analyses. (B) Effect of dexamethasone on OCT1 protein expression in primary human hepatocytes. Primary human hepatocytes (Batch Hep220624, Hep220633and LH46) were pre-cultivated in either Dex-supplemented Isom medium (+Dex medium) or Dex-free medium (-Dex medium). Hepatocytes were then treated for 48 h with RU486 (1 mM), dexamethasone (Dex; 100 or 500 nM), the combination of dexamethasone and RU486 or vehicle (0.1% DMSO, control). The cells were lysed, and the total cellu- lar lysates were resolved and underwent western blotting analysis. A rabbit polyclonal antibody against OCT1 (ab55916, Abcam, Cambridge, UK) was used. Numbers below the autoradiograms refer to the intensity of the bands as determined by densitometric analysis. Vehicle-treated samples are set to a value of 1
protein and no significant effect of the treatment on OCT1 protein expression in the cell line (Fig. 2C).
Based on the data, we can exclude any functional GR response element in the 1.7 kb promoter sequence of the OCT1 gene and we suppose that OCT1 might not be a target gene directly transactivated by GR.
Adenoviral-mediated transduction of HNF4a, PGC1a and C/EBPb increases the mRNA level from the OCT1 gene in Mz-Hep1 cells
In another experiments, we examined the effects of nu- clear receptors and LETFs on OCT1 mRNA expression
Fig. 2. Effects of dexamethasone on the OCT1 expression in the MZ-Hep1 and HepG2 cell lines. (A) MZ-Hep1 or HepG2 cells were treated with dexamethasone (Dex; 100 nM), mifepristone (RU486; 1 mM), the combination of dexamethasone and mifepristone or vehicle control (0.1%
DMSO) for 24 h. When indicated, cells were transfected with GRa expression construct (50 ng/105cells). Total RNA was isolated, and mRNA levels were analyzed by qRT-PCR. The effects of the compounds on OCT1 mRNA expression are presented as fold mRNA expression relative to control vehicle-treated cells (set to a value of 1). (B) Transient transfection gene reporter assays with the OCT1 (-1649/+102)-luc, pOCT1(-1458/+102)DHNF4aRE-luc, and pOCT1(-430/+102)-luc reporter constructs (100 ng per 48-well plate well) were performed in HepG2 cells. Cells were cotransfected with expression constructs for GRa (or pSG5 vectors, 100 ng per well). After 24 h cultivation, the cells were treated with Dex (100 nM) or vehicle (0.1% DMSO) for 24 h. After the incubation, firefly luciferase activity was analyzed together with Renilla lu- ciferase activity. The data are presented as the fold activation relative to vehicle-treated empty reporter construct-transfected cells (set to a value of 1). (C) Western blotting analyses of OCT1 expression in MZ-Hep1 cells. Cells were treated with dexamethasone (Dex; 100 nM) or ve- hicle control (0.1% DMSO) for 48 h. Protein expression analyses were performed with the rabbit polyclonal antibody against human OCT1 (SLC22A1, ab55916, Abcam) according to protocol described in Materials and Methods section
in MZ-Hep1 and HeG2 cells. We aimed to elucidate whether HNF4a was the only critical transcription fac- tor involved in OCT1 gene transactivation. We ob- served a significant increase of OCT1 mRNA in MZ- Hep1 cells infected with Ad-HNF4a, Ad-PGC1a and Ad-C/EBPb (Fig. 3A). We found out that OCT1 mRNA up-regulation has a dose-dependent relation-
ship with the amount of HNF4a, PGC1 and C/EBPb cDNA transduced into the MzHep1 cells by adenoviral infection (Fig. 3A). Infection with PGC1a alone also up-regulated OCT1 mRNA, which suggested coopera- tion between PGC1a and another nuclear receptor or transcription factor expressed in the Mz-Hep1 cells.
Nevertheless, no ligand-activated human nuclear re-
Fig. 3. Effects of adenoviral transduction/transfection of HNF4a, PGC1a and C/EBPb cDNA on OCT1 mRNA levels in MZ-Hep1 and HepG2 cell lines. (A) Mz-Hep1 cells were transduced with increasing doses of HNF4a (5-20 M.O.I.), PGC1a (1-8 M.O.I.) and C/EBPb (5-20 M.O.I.) and harvested 48 h later. OCT1 mRNA levels were determined by qRT-PCR analysis and normalized to the PBGD housekeeping gene. The data represent the fold increase above the control non-transduced cells. Two to four independent experiments were performed in triplicate. Data from the most representative experiments are shown. MZ-Hep1 (B) cells were transduced with optimal doses of HNF4a (10 M.O.I.), PGC1a (4 M.O.I.) and C/EBPb (10 M.O.I.). The cells were lysed after 48 h. The OCT1 mRNA levels were measured by qRT-PCR analysis and normal- ized to the PBGD housekeeping gene. The data are expressed relative to non-transduced cells and represent the mean ± SD from three inde- pendent experiments. * p < 0.05, statistically significant to control;_p < 0.01, statistically significant to HNF4a-transduced cells. (C) C/EBPb transfection activates OCT1(-1649/+102)-luc gene reporter construct in HepG2 cells. Cells were cotransfected with gene reporter construct (100 ng per 48-well plate well), expression construct for C/EBPb-LAP or HNF4a (or empty vector, 100 ng per well) and Renilla luciferase ex- pression construct pRL-TK for transfection normalization. After 24 h cultivation, firefly luciferase activity was analyzed together with Renilla lucife- rase activity. The data are presented as the fold activation relative to cells transfected with pGL4.10-luc and empty expression vectors (set to a value of 1)
ceptor has been identified so far to induce OCT1 gene expression. This phenomenon remains to be eluci- dated. In this study, other LETFs (such as HNF1a, HNF3g) and the PXR and CAR nuclear receptors were transiently expressed in HepG2 cells, and no signifi- cant effects on OCT1 mRNA up-regulation were ob- served (data not shown).
In the next experiments, we examined the possible synergy between the studied transcriptional factors by co-infection into Mz-Hep1 cells. We optimized the volumes of adenoviruses (10 M.O.I. for HNF4a, 4 M.O.I. for PGC1a and 10 M.O.I. for C/EBPb) as a compromise between high transduction response and low cytotoxic effect. The results showed a signifi- cant synergistic effect of the HNF4a/PGC1a combi- nation (10.5-fold increase for the combination vs.
4.5-fold for HNF4a alone and 2.1-fold increase for PGC1a alone) and a synergistic effect for the C/EBPb/ HNF4a combination (10.4-fold for the com- bination vs. 5.1-fold for C/EBPb alone) (Fig. 3B).
Finally, we examined if C/EBPb transfection of HepG2 cells stimulate OCT1 gene reporter construct in transient transfection gene reporter assay. We found clear effect of C/EBPb co-transfection on OCT1 pro- moter activation (Fig. 3C). In agreement with pub- lished data, we observed that the OCT1 (-1649/+102) gene promoter luciferase construct is responsive to co- transfection with HNF4a expression construct in HepG2 cells, although the cell line expresses high lev- els of HNF4a mRNA (Fig. 3C) [13, 23]. Our data now indicate that C/EBPb may also have a functional re- sponse element(s) within 1.7 kb of the OCT1 promoter sequence to transactivate expression of the gene.
Expression of OCT1 mRNA correlates with the expression of HNF4a and C/EBPb mRNAs
In the next series of experiments, we used a set of hu- man liver samples and investigated the correlation between OCT1 mRNA levels and those of HNF4a (Fig. 4A), C/EBPb (Fig. 4B) or PGC1a (Fig. 4C). We observed a statistically significant correlation be- tween the mRNA levels of OCT1 and HNF4a (Pear- son correlation coefficient r = 0.58; p < 0.001). The correlation between OCT1 and C/EBPb mRNAs was weaker (r = 0.33, p < 0.05). No significant correlation was observed (95% confidence interval -0.6266 to 0.4189) between OCT1 and PGC1a mRNAs. These findings indicate that HNF4a and C/EBPb are in- volved in OCT1 gene regulation in human hepato-
cytes. However, the expression of PGC1a does not seem to be the critical determinant of OCT1 mRNA expression in human hepatocytes.
Fig. 4. Correlation analysis of OCT1 mRNA expression relative to HNF4a (A), C/EBPb (B) and PGC1a (C) mRNA expression levels in liver samples. The mRNA levels from a set of 42 liver samples (14 samples for PGC1a) were measured by qRT-PCR analysis and normalized to the PBGD housekeeping gene. The data are ex- pressed relative to a pool of mRNAs from human liver tissues. Pear- son correlation coefficients (r) are provided
Glucocorticoid dexamethasone affects HNF4a, but not on C/EBPb and PGC1a, expression in primary human hepatocytes
Next, we investigated the effect of GR activation or in- hibition on C/EBPb and PGC1a mRNA expression in four primary human hepatocyte preparations. The ef- fect of GR activation on HNF4a mRNA have been de- scribed in our previous paper [30]. We found a statisti- cally significant (p < 0.01) effect of RU486 on PGC1a mRNA expression in +Dex medium (Fig. 5A). How- ever, Dex had no significant effect on PGC1a mRNA expression in primary hepatocytes maintained in the glucocorticoids-free -Dex medium (Fig. 5A). We also did not observe any effect of GR activation or inhibi- tion on C/EBPb mRNA expression (Fig. 5A).
In the western blotting experiments, we found that HNF4a protein expression was increased by a 24 or 48 h Dex treatment (100 nM) if the hepatocytes were originally cultivated in –Dex medium (Fig. 5B).
Finally, we did not observe any clear profile for PGC1a protein expression that would indicate any ef- fect of dexamethasone (100 nM) or RU486 on PGC1a protein expression in primary human hepato- cytes after 24 or 48 h (Fig. 5C). b-Actin, a reference protein used in each protein sample as a loading con- trol, has not been affected in any experiments.
Dexamethasone does not induce HNF4a gene in hepatocyte-derived tumor cell lines
In the last series of experiments, we used HepG2 and MZ-Hep1 cell lines and treated them with Dex (100 nM), RU486 (1 mM) or their combination. We observed that Dex did not induce HNF4a mRNA in these cell lines (Fig. 6A). Co-transfection of the cells with GR expression construct did not have any effect on OCT1 mRNA expression. Consistently, we did not observe any effect of Dex on the HNF4a-responsive gene reporter luciferase construct pGL3-B-3xApoCII- TK-luc, which was transfected into both the HepG2 and the MZ-Hep1 cells (Fig. 6B). Cotransfection of HNF4a expression construct into the MZ-Hep1 cells resulted in much more intensive activation of the HNF4a-responsive gene reporter luciferase construct than in case of HepG2 cells. This finding is in agree- ment with our previous observation indicating very low expression of HNF4a mRNA in MZ-Hep1 cells compared with HepG2 cells and primary human hepa- tocytes [13].
Finally, Dex treatment did not change HNF4a pro- tein expression in both cell lines (Fig. 6C).
These data suggest that HNF4a is not up-regulated by glucocorticoid receptor in the MZ-Hep1 and HepG2 cell lines, which results in a lack of Dex effect on OCT1 gene expression and transactivation. In addition, based on these data, we can exclude any functional GR response element in the 1.7 kb promoter sequence of the OCT1 gene and we suppose that OCT1 might not be a target gene directly transactivated by GR.
Discussion
In the current study, we demonstrate a significant ef- fect of dexamethasone on OCT1 mRNA and protein induction in primary human hepatocytes after 48 h of treatment, although OCT1 promoter gene reporter construct is not directly transactivated by dexametha- sone. We also confirmed that HNF4a is a critical fac- tor in OCT1 gene transactivation in primary human hepatocytes [23], which is induced by GR activation.
In addition, we discovered that C/EBPb contributes to transactivation of the OCT1 gene in hepatocyte tumor-derived cell lines and that OCT1 mRNA ex- pression significantly correlates with C/EBPb mRNA expression in human hepatocytes. Moreover, we show a synergistic effect between C/EBPb and HNF4a on OCT1 gene expression in hepatocyte tumor-derived cell lines (Fig. 7). Finally, we found that OCT1 is regulated only in normal hepatocytes but GR up- regulates neither OCT1 nor HNF4a in model hepato- carcinoma cell lines HepG2 and MZ-Hep1. This dis- crepancy may be connected either with expres- sion/downregulation of a tumor specific factor or acti- vation or inhibition of a signaling tumorigenic pathway that modulates activity of transcription fac- tors involved in OCT1 gene regulation.
OCT1 is a transporter of many endogenous and ex- ogenous compounds and is involved in the hepatic up- take of a number of agents used in current pharmaco- therapy [12]. The role of OCT1 in the distribution, elimination and therapeutic effect of small organic cations such as the biguanide antidiabetic drug met- formin has recently been clearly demonstrated in Oct1/2-/- knockout mice or in persons with reduced functioning of the OCT1 alleles R61C, G401S, 420del, or G465R [25, 26, 31]. Nevertheless, regula-
tion and liver-specific expression of the OCT1 gene remains incompletely understood.
There is accumulating evidence that the glucocorti- coid receptor is an upstream hormonal regulator that controls the expression and transcriptional activity of numerous hepatic nuclear receptors and transcription factors critical for gene regulation of xenobiotic-me-
tabolizing enzymes and drug transporters in the liver [5, 20, 30].
HNF4a was shown to be induced by glucocorti- coids in rodent livers, and its expression and tran- scriptional activity were demonstrated to be aug- mented by the glucocorticoid receptor in human hepa- tocytes [7, 14, 17, 18, 30]. Similarly, C/EBPb and its
Fig. 5. Effects of dexamethasone on HNF4a, PGC1a and C/EBPb mRNA expression in primary human hepatocytes. (A) Four primary human hepatocyte cultures were maintained for 16 h in standard medium (+Dex medium) or dexamethasone-free medium (-Dex medium). Hepato- cytes were subsequently treated for 24 h with dexamethasone (Dex; 100 nM), RU486 (1 mM), the combination of dexamethasone and RU486 or vehicle control (0.1% DMSO). Total RNA was isolated, and the mRNA levels for PGC1a and C/EBPb were analyzed by qRT-PCR. The effects of the compounds on target gene mRNA levels are presented as the fold mRNA expression relative to control vehicle-treated cells in +Dex me- dium (set to a value of 1). Western blotting analyses of HNF4a (B) and PGC1a (C) expression. Primary human hepatocytes were treated with dexamethasone (Dex; 100 nM), RU486 (1 mM), the combination of dexamethasone and mifepristone or vehicle control (0.1% DMSO) for 24 or 48 h in glucocorticoid-free (-Dex) medium. Protein expression analyses in primary human hepatocytes were performed with the rabbit anti- HNF4a polyclonal antibody and the goat anti-PGC1a polyclonal antibody according to protocol described in Materials and Methods section
LAP isoform were reported to be up-regulated after dexamethasone treatment of primary mouse hepato- cytes and hepatoma cell lines [1, 8, 33]. Finally, using a Profiler™ PCR Array, we found that the mRNA ex- pression of the peroxisome proliferator-activated re- ceptor g coactivator-1a (PGC-1a) may be up- regulated by dexamethasone in primary human hepa- tocytes [30]. In the current study, however, PGC1a protein and C/EBPb mRNA expression were not sig-
nificantly regulated by treatment with a GR agonist or the antagonist RU486 in primary human hepatocytes (Figs. 5A and 5C).
We only confirmed significant up-regulation of the HNF4a protein after 48 h-treatment with dexametha- sone (100 nM) in both commercial hepatocytes and hu- man hepatocytes isolated in our laboratory (Fig. 5B).
These results do not agree with previously published data [17], which found no HNF4a protein up-regulation
Fig. 6. Effects of dexamethasone on the HNF4a expression in the MZ-Hep1 and HepG2 cell lines. (A) MZ-Hep1 or HepG2 cells were treated with dexamethasone (Dex; 100 nM), mifepristone (RU486; 1 mM), the combination of dexamethasone and mifepristone or vehicle control (0.1%
DMSO) for 24 h. When indicated, cells were transfected with GRa expression construct (50 ng/105cells). Total RNA was isolated, and mRNA levels were analyzed by qRT-PCR. The effects of the compounds on HNF4a mRNA expression are presented as fold mRNA expression relative to control vehicle-treated cells (set to a value of 1). (B) Transient transfection gene reporter assays with the HNF4a-responsive pGL3-B- 3xApoCIII-TK-luc construct (100 ng per 48-well plate well) were performed in HepG2 or MZ-Hep1 cells. Cells were cotransfected with expres- sion constructs for GRa or HNF4a2 (or empty pcDNA3 or pSG5 vectors, 100 ng per well). After 24 h cultivation, the cells were treated with Dex (100 nM) or vehicle (0.1% DMSO) for 24 h. After the incubation, firefly luciferase activity was analyzed together with Renilla luciferase activity.
The data are presented as the fold activation relative to vehicle-treated mock-transfected cells (set to a value of 1). Western blotting analyses of HNF4a (C) expression in MZ-Hep1 or HepG2 cells. Cells were treated with dexamethasone (Dex; 100 nM) or vehicle control (0.1% DMSO) for 48 h. Protein expression analyses were performed with the rabbit anti-HNF4a polyclonal antibody according to protocol described in Materials and Methods section
after treatment of primary human hepatocyte cultures with dexamethasone. This discrepancy may be due to inter-individual variation in response to GR activation in HNF4a expression or due to different methods used.
In the next series of experiments, we observed that OCT1 mRNA is not up-regulated by Dex in the cell lines (Fig. 2A) and that the 1.7 kb promoter of the OCT1 gene is not activated by dexamethasone in a transient transfection gene reporter assay in the he- patocyte tumor HepG2 cells (Fig. 2B). Since dex- amethasone does not induce HNF4a in the cell lines (Fig. 6), these data altogether indicate that there is no functional GR response element within 1.7 kb of the 5´-flanking promoter region of the OCT1 gene. In ad- dition, the data confirm the hypothesis that OCT1 is indirectly regulated by GR receptor via HNF4a up- regulation in primary human hepatocytes. Neverthe- less, we cannot exclude a functional GR response ele- ment in flanking sequences or in other positions within the OCT1 (SLC22A1) gene chromosome loca- tion 6q25.3 and mediating direct transactivation of the OCT1 gene by the glucocorticoid receptor.
Differential effect of GR activation on OCT1 gene expression between tumor cell lines and normal pri- mary human hepatocytes might be due to epigenetic silencing of OCT1 in hepatocellular carcinoma de- scribed by Scheffeler et al. [24]. They found increased CpG methylation of the OCT1 promoter in hepatocel- lular carcinoma in comparison with normal hepato- cytes. We can therefore suppose that OCT1 promoter may be methylated in HepG2 and MZ-Hep1 cells
which suppress transactivation by GR. Nevertheless, direct demonstration of epigenetic regulation of OCT1 gene expression in hepatocellular carcinoma cell lines has not been reported.
We also found that the 1.7 kb promoter construct of the OCT1 gene is activated after C/EBPb-LAP overex- pression in HepG2 cells (Fig. 3C). These data indicate the presence of a C/EBPb binding site(s) within -1649 to +102 sequence of OCT1 gene. Using in silico analy- sis (TESS:Transcription Element Search System Web tool), we did find two hypothetical C/EBPb response element sequences within this region located about 0.7 and 0.2 kilobases upstream of active transcription start site. Further assays in our laboratories with gene re- porter constructs containing mutations or progressive 5’-deletions should delineate functional binding sites of C/EBPb in OCT1 promoter sequence.
HNF4a, PGC1a and C/EBPb are factors involved in transcriptional regulation of basic hepatic metabolic path- ways such as bile acids metabolism and glucose homeo- stasis. HNF4a itself controls the development of the he- patic epithelium and phenotype and liver morphogenesis [19]. HNF4a can also rescue the expression of liver genes in dedifferentiated hepatoma cell lines [24, 28, 29].
Therefore, the question arises whether there is any un- known endogenous function of OCT1 related to interme- diary metabolism that has not yet been discovered.
We can conclude that the SLC22A1 gene encoding the OCT1 hepatic transporter is transactivated by glu- cocorticoids in primary human hepatocytes, but not in hepatocyte-derived tumor cell lines. HNF4a but not Regulation of SLC22A1 gene in primary human hepatocytes
exon exon
SLC22A1
HNF4 dexamethasone
exon exon
HNF4
C/EBP
HNF4
OCT1
nucleus GR GR Fig. 7. Regulation of SLC22A1 gene in
primary human hepatocytes
the C/EBPb and PGC1a transcription factors are sig- nificantly regulated by physiological concentrations of glucocorticoids in primary human hepatocytes. We also demonstrate that C/EBPb is involved in regula- tion of OCT1 gene expression in primary human he- patocytes (Fig. 7). Further studies are needed to eluci- date the effect of glucocorticoids on additional HNF4a-controlled genes as well as on accumulation of OCT1 substrates in human liver.
Acknowledgments:
We gratefully acknowledge Jana Mandikova for her technical assistance. We thank Dr. Palvilo for the GR expression construct.
This work was supported by the Czech Scientific Agency (grants No. 303/12/G163 and 303/12/0472 to P.P.) and by grant PI 10/00194 from Fondo de Investigación Sanitaria (FIS, Instituto de Salud Carlos III, Ministerio de Economía y Competitividad, Spain).
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Received: January 28, 2013; in the revised form: May 15, 2013;
accepted: June 7, 2013.
