Glucocorticoid receptor regulates organic cation

transporter 1 (OCT1, SLC22A1) expression via

HNF4� upregulation in primary human hepatocytes

Alice Rulcova1*, Lucie Krausova1*, Tomas Smutny1, Radim Vrzal2,

Zdenek Dvorak2, Ramiro Jover3,4, Petr Pavek1

�Department of Pharmacology and Toxicology, Faculty of Pharmacy in Hradec Kralove, Charles University

in Prague, Heyrovskeho 1203, Hradec Kralove, CZ-500 05, Czech Republic

�Department of Cell Biology and Genetics, Faculty of Science, Palacky University in Olomouc, Slechtitelu 11,

Olomouc, CZ-783 71, Czech Republic

�Unidad Mixta Hepatología Experimental, Hospital La Fe & University of Valencia (Dep. Biochemistry and

Molecular Biology), Av. Campanar, 21, 46009 Valencia, Spain

�CIBERehd, 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-4�

(HNF4�). HNF4� 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 HNF4� 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 HNF4� 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 HNF4�, CCAAT/enhancer binding proteins � (C/EBP�) and peroxisome proliferator-activated recep-

tor-� coactivator 1� (PGC1�) 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/EBP� and HNF4� mRNAs expression and that C/EBP�

co-transfection stimulates OCT1 gene reporter construct in HepG2 cells. Nevertheless, neither C/EBP� nor PGC1� were up-

regulated in human hepatocytes by dexamethasone.

Conclusion: We can conclude that GR-induced expression of HNF4� 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, HNF4�, C/EBP�, gene regulation, hepatocyte

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* 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 HNF4� nuclear receptor via two DR-2 sites [23].

HNF4� 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, HNF4�

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 HNF4� gene causes

a monogenic form of early-onset type 2 diabetes

(maturity-onset diabetes of the young, MODY1) [34].

HNF4� belongs, together with other transcription

factors such as CCAAT/enhancer binding proteins �

and � (C/EBP� and C/EBP�) and hepatocyte nuclear

factor 1� (HNF1�) and 3� (HNF3�, 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 HNF4�-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 HNF4�

silencing [11]. Thus, we can suppose that stimulation

of HNF4� 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 HNF4� in human

and rodent hepatocytes. We found that dexametha-

sone significantly up-regulated the mRNA of HNF4�

in primary human hepatocytes. The HNF4� mRNA

induction by dexamethasone was totally inhibited by

the glucocorticoid receptor antagonist RU486 [30].

HNF4� 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 HNF4� protein up-regulation in hu-

man hepatocytes. Nevertheless, they discovered that

dexamethasone increased HNF4� 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 HNF4� 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 HNF4� and

C/EBP�-LAP were developed as described earlier [10,

13]. The adenoviral vector for the expression of PGC1�

was kindly provided by Dr. P. Puigserver (Johns Hop-

kins University School of Medicine, Baltimore, MD,

������������� �� ����� ����� ��� ��������� 1323

GR regulates OCT1 expression in human hepatocytes����� ����� �� ��

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 HNF4� 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 HNF4�. pOCT1(�1458/+102)

�HNF4�RE-luc construct lacks the first HNF4� re-

sponse element (A) critical for OCT1 promoter trans-

activation with HNF4�. 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-hGR� expression construct

(a gift from Dr. Palvimo, University of Helsinki, Hel-

sinki, Finland) before the treatment with dexametha-

sone [15]. Expression construct for C/EBP�-LAP has

been described in our previous paper [10]. A chimeric

luciferase reporter construct, containing three in-

tandem copies of the HNF4� 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/EBP� or

PGC1� 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 HNF4�, C/EBP� or PGC1� 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 MgCl� 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-

1324 ������������� �� ����� ����� ��� ���������

producibility of the measurements was assessed by

conducting triplicate reactions.

Western blotting analysis

Immunodetection of OCT1, HNF4� and PGC1� 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 MgCl�, 100 mM KCl, pH

7.9) including dithiothreitol and protease inhibitors.

After electrophoretic resolution and transfer onto

membranes, primary rabbit polyclonal antibodies

against OCT1, HNF4�, and PGC1� were used. The

rabbit polyclonal antibody against human OCT1

(SLC22A1, ab55916) was obtained from Abcam,

Cambridge, UK. Antibodies against HNF4� (rabbit

polyclonal; sc-8987, H-171) and PGC-1� (goat poly-

clonal; sc-8987, H-171) were purchased from Santa

Cruz Biotechnology (Santa Cruz, USA). �-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 �M, 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 �M) or their combina-

tion. We found that Dex does not induce OCT1

������������� �� ����� ����� ��� ��������� 1325

GR regulates OCT1 expression in human hepatocytes����� ����� �� ��

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’

HNF4�Forward

Reverse

5’-GCC TAC CTC AAA GCC ATC AT-3’

5’-GAC CCT CCC AGC AGC ATC TC-3’

PGC1�Forward

Reverse

5’-AAT GTG TCT CCT TCT TGT TCT T-3’

5’-GGT GTC TGT AGT GGC TTG TTG A-3’

C/EBP�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

HNF4� 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

1326 ������������� �� ����� ����� ��� ���������

Fig. 1. Effects of dexamethasone on OCT1 mRNA and protein expression in primary human hepatocytes. (A) Four primary human hepatocytecultures LH155, 157, 164 and 167 were maintained for 16 h in standard Isom medium (+Dex medium) or dexamethasone-free medium (�Dexmedium) and subsequently treated for 24 h with RU486 (1 �M), dexamethasone (Dex; 100 nM), the combination of dexamethasone and RU486or vehicle control (0.1% DMSO). Total RNA was isolated and mRNA levels were analyzed using qRT-PCR. The effects of compounds on OCT1gene mRNA levels are presented as fold mRNA expression relative to control vehicle-treated cells cultivated in +Dex medium (set to a value of1). Data are presented as the means of three qRT-PCR analyses. (B) Effect of dexamethasone on OCT1 protein expression in primary humanhepatocytes. Primary human hepatocytes (Batch Hep220624, Hep220633and LH46) were pre-cultivated in either Dex-supplemented Isommedium (+Dex medium) or Dex-free medium (�Dex medium). Hepatocytes were then treated for 48 h with RU486 (1 �M), 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-treatedsamples 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 HNF4�,

PGC1� and C/EBP� 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

������������� �� ����� ����� ��� ��������� 1327

GR regulates OCT1 expression in human hepatocytes����� ����� �� ��

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 withdexamethasone (Dex; 100 nM), mifepristone (RU486; 1 �M), the combination of dexamethasone and mifepristone or vehicle control (0.1%DMSO) for 24 h. When indicated, cells were transfected with GR� expression construct (50 ng/10� cells). Total RNA was isolated, and mRNAlevels were analyzed by qRT-PCR. The effects of the compounds on OCT1 mRNA expression are presented as fold mRNA expression relativeto 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)�HNF4�RE-luc, and pOCT1(�430/+102)-luc reporter constructs (100 ng per 48-well plate well) were performed in HepG2cells. Cells were cotransfected with expression constructs for GR� (or pSG5 vectors, 100 ng per well). After 24 h cultivation, the cells weretreated 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 toa 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 HNF4� 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-HNF4�, Ad-PGC1� and

Ad-C/EBP� (Fig. 3A). We found out that OCT1

mRNA up-regulation has a dose-dependent relation-

ship with the amount of HNF4�, PGC1 and C/EBP�

cDNA transduced into the MzHep1 cells by adenoviral

infection (Fig. 3A). Infection with PGC1� alone also

up-regulated OCT1 mRNA, which suggested coopera-

tion between PGC1� and another nuclear receptor or

transcription factor expressed in the Mz-Hep1 cells.

Nevertheless, no ligand-activated human nuclear re-

1328 ������������� �� ����� ����� ��� ���������

Fig. 3. Effects of adenoviral transduction/transfection of HNF4�, PGC1� and C/EBP� cDNA on OCT1 mRNA levels in MZ-Hep1 and HepG2 celllines. (A) Mz-Hep1 cells were transduced with increasing doses of HNF4� (5-20 M.O.I.), PGC1� (1�8 M.O.I.) and C/EBP� (5-20 M.O.I.) andharvested 48 h later. OCT1 mRNA levels were determined by qRT-PCR analysis and normalized to the PBGD housekeeping gene. The datarepresent the fold increase above the control non-transduced cells. Two to four independent experiments were performed in triplicate. Datafrom the most representative experiments are shown. MZ-Hep1 (B) cells were transduced with optimal doses of HNF4� (10 M.O.I.), PGC1�(4 M.O.I.) and C/EBP� (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 HNF4�-transduced cells. (C) C/EBP�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/EBP�-LAP or HNF4� (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 toa 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 HNF1�,

HNF3�) 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 HNF4�,

4 M.O.I. for PGC1� and 10 M.O.I. for C/EBP�) as

a compromise between high transduction response

and low cytotoxic effect. The results showed a signifi-

cant synergistic effect of the HNF4�/PGC1� combi-

nation (10.5-fold increase for the combination vs.

4.5-fold for HNF4� alone and 2.1-fold increase for

PGC1� alone) and a synergistic effect for the

C/EBP�/ HNF4� combination (10.4-fold for the com-

bination vs. 5.1-fold for C/EBP� alone) (Fig. 3B).

Finally, we examined if C/EBP� transfection of

HepG2 cells stimulate OCT1 gene reporter construct in

transient transfection gene reporter assay. We found

clear effect of C/EBP� 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 HNF4� expression construct in

HepG2 cells, although the cell line expresses high lev-

els of HNF4� mRNA (Fig. 3C) [13, 23]. Our data now

indicate that C/EBP� 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 HNF4� and C/EBP� 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 HNF4�

(Fig. 4A), C/EBP� (Fig. 4B) or PGC1� (Fig. 4C). We

observed a statistically significant correlation be-

tween the mRNA levels of OCT1 and HNF4� (Pear-

son correlation coefficient r = 0.58; p < 0.001). The

correlation between OCT1 and C/EBP� 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 PGC1� mRNAs. These

findings indicate that HNF4� and C/EBP� are in-

volved in OCT1 gene regulation in human hepato-

cytes. However, the expression of PGC1� does not

seem to be the critical determinant of OCT1 mRNA

expression in human hepatocytes.

������������� �� ����� ����� ��� ��������� 1329

GR regulates OCT1 expression in human hepatocytes����� ����� �� ��

Fig. 4. Correlation analysis of OCT1 mRNA expression relative toHNF4� (A), C/EBP� (B) and PGC1� (C) mRNA expression levelsin liver samples. The mRNA levels from a set of 42 liver samples(14 samples for PGC1�) were measured by qRT-PCR analysis andnormalized 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 HNF4�,

but not on C/EBP� and PGC1�, expression in

primary human hepatocytes

Next, we investigated the effect of GR activation or in-

hibition on C/EBP� and PGC1� mRNA expression in

four primary human hepatocyte preparations. The ef-

fect of GR activation on HNF4� mRNA have been de-

scribed in our previous paper [30]. We found a statisti-

cally significant (p < 0.01) effect of RU486 on PGC1�

mRNA expression in +Dex medium (Fig. 5A). How-

ever, Dex had no significant effect on PGC1� 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/EBP� mRNA expression (Fig. 5A).

In the western blotting experiments, we found that

HNF4� 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

PGC1� protein expression that would indicate any ef-

fect of dexamethasone (100 nM) or RU486 on

PGC1� protein expression in primary human hepato-

cytes after 24 or 48 h (Fig. 5C). �-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 HNF4� 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 �M) or their combination. We

observed that Dex did not induce HNF4� 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 HNF4�-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

HNF4� expression construct into the MZ-Hep1 cells

resulted in much more intensive activation of the

HNF4�-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 HNF4� mRNA in MZ-Hep1 cells

compared with HepG2 cells and primary human hepa-

tocytes [13].

Finally, Dex treatment did not change HNF4� pro-

tein expression in both cell lines (Fig. 6C).

These data suggest that HNF4� 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 HNF4� 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/EBP� contributes

to transactivation of the OCT1 gene in hepatocyte

tumor-derived cell lines and that OCT1 mRNA ex-

pression significantly correlates with C/EBP� mRNA

expression in human hepatocytes. Moreover, we show

a synergistic effect between C/EBP� and HNF4� 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 HNF4� 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-

1330 ������������� �� ����� ����� ��� ���������

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].

HNF4� 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/EBP� and its

������������� �� ����� ����� ��� ��������� 1331

GR regulates OCT1 expression in human hepatocytes����� ����� �� ��

Fig. 5. Effects of dexamethasone on HNF4�, PGC1� and C/EBP� mRNA expression in primary human hepatocytes. (A) Four primary humanhepatocyte 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 �M), the combination of dexamethasone and RU486 orvehicle control (0.1% DMSO). Total RNA was isolated, and the mRNA levels for PGC1� and C/EBP� were analyzed by qRT-PCR. The effects ofthe 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 HNF4� (B) and PGC1� (C) expression. Primary human hepatocytes were treated withdexamethasone (Dex; 100 nM), RU486 (1 �M), the combination of dexamethasone and mifepristone or vehicle control (0.1% DMSO) for 24 or48 h in glucocorticoid-free (�Dex) medium. Protein expression analyses in primary human hepatocytes were performed with the rabbit anti-HNF4� polyclonal antibody and the goat anti-PGC1� 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 � coactivator-1� (PGC-1�) may be up-

regulated by dexamethasone in primary human hepa-

tocytes [30]. In the current study, however, PGC1�

protein and C/EBP� 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

HNF4� 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 HNF4� protein up-regulation

1332 ������������� �� ����� ����� ��� ���������

Fig. 6. Effects of dexamethasone on the HNF4� expression in the MZ-Hep1 and HepG2 cell lines. (A) MZ-Hep1 or HepG2 cells were treatedwith dexamethasone (Dex; 100 nM), mifepristone (RU486; 1 �M), the combination of dexamethasone and mifepristone or vehicle control (0.1%DMSO) for 24 h. When indicated, cells were transfected with GR� expression construct (50 ng/10� cells). Total RNA was isolated, and mRNAlevels were analyzed by qRT-PCR. The effects of the compounds on HNF4� mRNA expression are presented as fold mRNA expression relativeto control vehicle-treated cells (set to a value of 1). (B) Transient transfection gene reporter assays with the HNF4�-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 GR� or HNF4�2 (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 ofHNF4� (C) expression in MZ-Hep1 or HepG2 cells. Cells were treated with dexamethasone (Dex; 100 nM) or vehicle control (0.1% DMSO) for48 h. Protein expression analyses were performed with the rabbit anti-HNF4� polyclonal antibody according to protocol described in Materialsand 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 HNF4� 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 HNF4� 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 HNF4� 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/EBP�-LAP overex-

pression in HepG2 cells (Fig. 3C). These data indicate

the presence of a C/EBP� 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/EBP� 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/EBP� in OCT1 promoter sequence.

HNF4�, PGC1� and C/EBP� are factors involved in

transcriptional regulation of basic hepatic metabolic path-

ways such as bile acids metabolism and glucose homeo-

stasis. HNF4� itself controls the development of the he-

patic epithelium and phenotype and liver morphogenesis

[19]. HNF4� 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. HNF4� but not

������������� �� ����� ����� ��� ��������� 1333

GR regulates OCT1 expression in human hepatocytes����� ����� �� ��

Regulation of SLC22A1 gene in primary human hepatocytes

exon exon

SLC22A1HNF4

dexamethasone

exon exon

HNF4

C/EBP

HNF4

OCT1

nucleus

GRGR

Fig. 7. Regulation of SLC22A1 gene inprimary human hepatocytes

the C/EBP� and PGC1� transcription factors are sig-

nificantly regulated by physiological concentrations

of glucocorticoids in primary human hepatocytes. We

also demonstrate that C/EBP� 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

HNF4�-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.

������������� �� ����� ����� ��� ��������� 1335

GR regulates OCT1 expression in human hepatocytes����� ����� �� ��