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To analyse systematically the rat Bsep promoter and to investigate the regulation of Bsep transcription by bile acids, drugs and endogenous compounds we identified the 5¢ flanking region o

Functional analysis of the rat bile salt export pump gene promoter

Regulation by bile acids, drugs and endogenous compounds

Thomas Gerloff1, Andreas Geier2, Ivar Roots1, Peter J Meier3and Carsten Gartung2

1

Institute of Clinical Pharmacology, Charite´ University Medical Center, Humboldt University, Berlin, Germany;

2

Department of Internal Medicine, Aachen University of Technology, Aachen, Germany;3Division of Clinical Pharmacology and Toxicology, Department of Medicine, University Hospital, Zurich, Switzerland

The 5¢ flanking region of the bile salt export pump (Bsep)

gene was systematically analysed to provide the basis for

understanding the mechanisms which regulate Bsep

tran-scription In addition substrates and drugs were investigated

for their ability to alter Bsep promoter activity Bsep

pro-moter function was restricted to hepatocyte derived HepG2

cells The 5¢ deletional analysis revealed a biphasic shape of

reporter gene activities, indicating a suppressive element

between nucleotides)800 and )512 Two consensus sites for

the farnesoid X receptor (FXR) were located at nucleotides

)473 and )64 The latter was characterized as functionally

active in bile acid-mediated feed-back regulation of Bsep

transcription Bsep promoter activity was reduced by rifampin and b-estradiol The anti-estrogen tamoxifen stimulated promoter activity Dexamethasone, hydrocorti-sone and phenobarbital had no effect on Bsep promoter activity In conclusion, the data suggest that transcriptional regulation of the Bsep gene can be modulated by a number of endogenous compounds and xenobiotics FXR was a major regulatory factor, mediating bile acid feed-back stimulation

of Bsep transcription

Keywords: bile flow; drug-induced cholestasis; transcrip-tional regulation

Bile secretion by vertebrate liver is caused by the continuous

vectorial excretion of bile acids and other osmotically active

substrates across the canalicular pole of hepatocytes Bile

acid-dependent bile flow provides the major driving force for

the generation and maintenance of liver excretory processes

and is therefore essential for proper hepatic clearance of

endogenous compounds and xenobiotics Canalicular

secre-tion of bile acids is predominantly mediated by the

transmembrane transporter system BSEP (human)/Bsep

(rodents) [1] Rat Bsep belongs to the superfamily of

ATP-binding cassette (ABC) transporters and is closely related to

P-glycoprotein, the gene product of Mdr1a/1b The Bsep

gene encodes a 160-kDa polypeptide which is highly, and

almost exclusively, expressed in the canalicular membrane of

hepatocytes Impairment of the Bsep transporter system

results in cholestasis due either to inherited mutations of its

gene [2], or secondary to dysfunction caused by biliary

obstruction, xenobiotics or systemic inflammation

Regulation of protein expression of hepatic transport

systems plays an important role in the production of bile in

normal and diseased liver Major goals in the regulation of hepatocellular transporters are to prevent intracellular accumulation of toxic bile acids and to maintain biliary flow for ongoing hepatic clearance A differential expression

of the basolateral bile acid uptake system Na+-taurocholate cotransporting polypeptide (Ntcp) and the canalicular Bsep could clearly be demonstrated in animal models of choles-tasis and liver regeneration [3,4] Whereas Ntcp was down-regulated in these models, Bsep expression was sustained, thus protecting hepatocytes from damage caused by toxic bile acids and metabolites Observations from farnesoid X receptor (FXR)-deficient mice [5] fed a diet of cholic acid (CA) demonstrated that FXR is a critical transcription factor that controls differential expression of the key liver cell basolateral and canalicular bile acid transporters Ntcp and Bsep While CA feeding resulted in a large increase of Bsep mRNA levels in the livers of FXR wild-type mice, no increase was observed in FXR-null mice [5] In contrast Ntcp mRNA was down-regulated following CA feeding but remained unchanged in FXR-deficient mice Recent studies

on the human BSEP and rat Ntcp promoters support this concept [6,7]

Treatment with drugs frequently results in impairment

of liver function [8] A variety of mechanisms have been described to cause drug-induced cholestasis, including decreased hepatocellular bile secretion [9] Steroid hormones like estradiol have been demonstrated to down-regulate Ntcp and Bsep mRNA levels [3] or to inhibit Bsep transport function [10] However, effects of drugs on Bsep promoter function have not been studied yet

To analyse systematically the rat Bsep promoter and to investigate the regulation of Bsep transcription by bile acids, drugs and endogenous compounds we identified the 5¢ flanking region of the rat Bsep gene and analysed its nucleotide sequence with respect to putative transcription

Correspondence to T Gerloff, Institute of Clinical Pharmacology,

Charite´, University Medical Center, Humboldt University Berlin,

Schumannstrasse 20/21, D-10098 Berlin, Germany.

Fax: +49 30 94063329, Tel.: +49 30 94062845,

E-mail: thomas.gerloff@gmx.de

Abbreviations: Bsep, bile salt export pump; ABC, ATP-binding

cassette; FXR, farnesoid X receptor; Ntcp, Na+taurocholate

cotransporting polypeptide; CA, cholic acid; FXRE, FXR response

element; TC, taurocholic acid, TUDCA, tauroursodeoxycholic acid;

CDCA, chenodeoxycholic acid; DCA, deoxycholic acid;

LCA, lithocholic acid; OATP: organic anion transporting

polypeptide; RXR, retinoid X receptor.

(Received 9 April 2002, accepted 30 May 2002)

factor binding sites We determined the minimal Bsep

promoter region capable to mediate basal Bsep expression

and provide evidence for a liver-specific function of the Bsep

promoter Our data demonstrate FXR-mediated bile acid

feed-back regulation of Bsep promoter activity in HepG2

cells transfected with a Bsep-luciferase-reporter gene

con-struct, without any cotransfection of FXR expression

plasmids, as in a previous report [6] Furthermore the

present study indicates the ability of drugs and endogenous

compounds to affect Bsep transcription, thus altering

hepatic bile flow and clearance

M A T E R I A L S A N D M E T H O D S

Genomic cloning and sequence analysis of the 5¢ flanking

region

We amplified rat genomic DNA and Bsep cDNA by PCR

using the oligonucleotide primers 5¢-AACTGTTCTGGT

GTGGATTCC-3¢ and 5¢-ATAGAAGATCTCTTGGTC

CTG-3¢ designed from the known rat Bsep cDNA clone [1]

As the two PCR products had the same length these

oligonucleotide primers were used for screening a rat P1

genomic DNA library (Genome Systems) by PCR Each

strand of the 5¢ flanking region of the Bsep gene was directly

sequenced (Epidauros, Bernried, Germany) by automated

sequencing The sequence was analysed for putative

cis-acting regulatory elements by using the transcription

factor databaseTRANSFAC4.0 (MatInspector V2.2, http://

transfac.gbf.de)

Construction of plasmids

A series of 5¢ deletions of the flanking region ranging from

nucleotides )1453 to )27 of upstream sequence and

extending downstream to nucleotide +80 were created by

PCR using a genomic P1 Bsep clone as a template The

forward primers were designed with a 5¢ flanking MluI

restriction site and the reverse primers contained a 5¢

flank-ing BglII site The PCR amplicons were cloned into the

EcoRV site of the pMOSBlue vector (Amersham Pharmacia

Biotech) and subsequently excised with MluI and BglII The

DNA fragments were then cloned directionally into the

MluI–BglII sites of the pGL3-Basic (Promega) luciferase

reporter gene vector The plasmid constructs were verified

by sequencing Mutations of the FXR response element

(FXRE) adjacent to the TATA box in the rat Bsep

promoter were generated using the QuickchangeTM

Site-Directed Mutagenesis Kit (Stratagene) The deletion

plas-mid extending to nucleotide)126 served as a template An

antisense (5¢-CACTGTTTGCTTATATTTCAATGGAA

TAAAGTCCAGCTCTAGC-3¢; exchanged bases in bold)

and sense (5¢-GCTAGAGCTGGACTTTATTCCATT

GAAATA-TAAGCAAACAGTG-3¢) oligonucleotide of

the IR-1 element was used in a temperature cycling reaction

as described in the manufacturer’s protocol to produce the

mutated plasmid m-126

Cell culture, transient transfections and reporter gene

assays

Human hepatoblastoma HepG2 (HB-8065, ATCC), colon

carcinoma CaCo2 (ACC169, DSMZ), Madin–Darby

canine kidney (MDCK; Dr Birchmeier, Max Delbru¨ck Center of Molecular Medicine, Berlin, Germany) and mouse fibroblast NIH 3T3 cells (Dr Blankenstein, Max Delbru¨ck Center of Molecular Medicine, Berlin, Germany) were cultured in Dulbecco’s modified Eagle medium containing 10% fetal bovine serum, 1% nonessential amino acids, 1 mmolÆL)1 sodium pyruvate, and 2 mmolÆL)1 glutamine Cells were transferred to six-well plates at 50–60% confluency and incubated at 37C 24 h prior to transient transfections Using TfxTM-20 (Promega) as cationic liposome 2.7 lg of reporter gene construct and 0.3 lg of the control reporter gene plasmid pRL-TK (Promega) were cotransfected The DNA–liposome mixture was removed after 2 h, and cells were incubated with Dulbecco’s modified Eagle medium containing 10% fetal bovine serum for an additional 48 h Cells were lysed, and cell extracts were assayed for luciferase activities in a Turner Designs TD-20/20 luminometer (Promega) using the Dual luciferase assay system (Promega) Relative reporter gene activities were expressed as the ratio of the firefly luciferase activity (reporter) and the renilla luciferase (transfection control) activity

Mapping of the transcriptional start site Two 5¢ RACE products were isolated from 1 lg total RNA from rat liver using gene-specific oligonucleotide primers corresponding to nucleotides 29–56 and 150–177 of the Bsep cDNA sequence [1] and subcloned into the pMosBlue vector (Amersham Pharmacia Biotech) Based on the sequence of the 5¢ RACE products an antisense oligonucle-otide extending 100 bp from nucleoligonucle-otide 62 of the Bsep cDNA was radiolabeled at the 5¢end with [c-32P]ATP (Ambion, AMS Biotechnology, Germany) to perform an S1-nuclease assay for the exact localization of the transcrip-tion initiatranscrip-tion site Total RNA from rat liver (100 lg) was hybridized with the labelled antisense probe at 42C for 2 h (Ambion, Germany) and subsequently digested with S1 nuclease for 30 min at 37C The remaining DNA–RNA hybridized fragments were electrophoresed on a 7% acryl-amide sequencing gel Sequencing reactions of the M13mp18 cloning vector were run in parallel and used as

a size marker After transferring the gel to 3 MM Whatman paper the dried gel was scanned using a phosphorimager (Raytest, Germany)

EMSA Preparation of nuclear extracts and EMSAs were performed

as described previously [11] Protein concentrations were determined according to Bradford [12] Nuclear extracts (5–10 lg protein) were incubated on ice for 30 min with a specific 32P-end-labeled oligonucleotide probe (2· 104c.p.m.) in a 20-lL reaction containing 8 lL water,

4 lL 5· binding buffer (25 mMHepes pH 7.6, 50 mMKCl, 0.5 mMdithiothreitol, 5 mMMgCl2, 0.5 mMEDTA, 10% glycerol) and 2 lg poly(dI-dC)-poly(dI-dC) (Amersham) For competition assays, 100-fold molar excess of specific unlabeled over labeled oligonucleotides were added to the binding reaction Samples were electrophoresed through a nondenaturing 6% polyacrylamide gel A double-stranded oligonucleotide containing the putative FXRE of the Bsep promoter sequence was used (sense strand sequence

5¢-GACTTTAGGCCATTGACCTATAAG-3¢) For

su-pershift experiments nuclear extracts were preincubated

for 30 min on ice with 1 lg of polyclonal antibodies (Santa

Cruz) either against FXRa, RXRa or both prior to addition

of the labelled oligonucleotide probe

Statistical analysis

All values are given as mean ± SD of triplicate

transfec-tions Student’s t-test was used to compare promoter

activities with controls P values < 0.05 were considered

to be statistically significant

R E S U L T S

Determination of the transcriptional start site

of theBsep gene

The transcription initiation site of the Bsep gene was located

by both 5¢ RACE amplification and S1 nuclease digestion

Two oligonucleotides corresponding to nucleotides 29–56

and 150–177 of the published Bsep cDNA [1] were used for

RACE Sequencing of the RACE products revealed that the

amplified 5¢ flanking regions started at nucleotide 6 and

nucleotide 8 of the cDNA sequence, respectively To exactly

map the transcription start site an oligonucleotide extending

100 bp from nucleotide 62 of the Bsep cDNA and including

the start region of the RACE products was subsequently

used in an S1 nuclease digestion assay Two protected

fragments of 73 and 64 bp were observed with total RNA

from rat liver (Fig 1) The larger fragment gave the

strongest signal intensity and was thus used to designate

nucleotide +1 in the Bsep promoter sequence No protected

fragments were identified using yeast tRNA as template

(data not shown)

Analysis of the 5¢ flanking region sequence

of theBsep gene

A total of 2488 bp upstream from the 5¢-end of the Bsep

cDNA was sequenced in both directions; 1583 bp are

shown in Fig 2 Identity with the Bsep cDNA sequence

begins at nucleotide +96 Several consensus matches for

potential transcription factor binding sites were identified

by searching the TRANSFAC 4.0 transcription

factor-binding site database (MATINSPECTOR V2.1) General DNA

elements containing motifs for a TATA box at nucleotide

)52, a CAAT box at nucleotide )66, multiple octamer

binding sites (nucleotides )708, )684, )667, )417, )396

and)197), and several NF1 sites (nucleotides )407, )237,

)211 and )73) could be detected Multiple binding sites

for the liver enriched transcription factor HNF3b were

found at nucleotides )718, )685, )661, )632 and )567

Interestingly there was only one HNF1 site (nucleotide

)839), and no consensus elements were found for the liver

enriched factor HNF4 Multiple AP1 sites are noted at

nucleotides )468, )447, )347, )338, )277, )243, )169,

)98 and ) 59 Three consensus motifs for C/EBP-b

binding elements are located at nucleotides )200, )582

and)757 Strikingly two binding sites for the

FXR/9-cis-retinoic acid receptor heterodimer at nucleotides)64 and

)473 could be identified These motifs are comprised of

two inverted repeats separated by one nucleotide (IR-1)

and have recently been demonstrated to function as bile acid responsive elements [13]

5¢ Deletional analysis of the Bsep promoter

in transfected HepG2 cells The 5¢ flanking regions of the rat Bsep promoter capable

of conferring basal activity was assessed by a series of deletion constructs cloned into the pGL3–luciferase reporter plasmid The Bsep promoter sequence inserted

in reverse orientation served as a negative control HepG2 cells were transfected with the reporter-plasmids and luciferase activities were determined relative to the level

of renilla luciferase cotransfected by the expression vector pRL-TK (Fig 3) Luciferase activity with the longest construct (p-1453) was set 100% Removal of the region between nucleotides )1453 and )800 resulted in a dramatic reduction of promoter activity by 90% How-ever, further deletion of the sequence to nucleotide )187 increased the activity to a maximum of 170% At

Fig 1 Determination of the transcriptional start site In a nuclease protection assay, an antisense oligonucleotide extending 100 bp from nucleotide 62 of the Bsep cDNA was hybridized with total RNA from rat liver and subsequently digested by S1 nuclease The major pro-tected fragment displayed in lane S (arrow) had a size of 73 bp The minor protected fragment was 64 bp in length Lanes A, C, G and T are sequence reactions of the M13mp18 cloning vector used as a size marker.

nucleotide )126 the luciferase activity reached nearly the

same levels as the p-1453 construct When the deletion

was extended further to nucleotide )27, the values

dropped to basal levels obtained with the promoterless

pGL3-Basic vector (data not shown) or with the construct

in reverse orientation (Fig 3) The minimal element

maintaining full promoter activity was identified as the

construct extending from nucleotide )126 to nucleotide

+80

Functional analysis of the Bsep promoter in cell lines

We investigated the tissue specificity of the Bsep

promo-ter activity by transfecting several nonhepatic cell lines

with the reporter constructs p-1453, p-187, p-126 and

p+80–1453 In contrast with HepG2 cells, no luciferase

reporter gene activation was observed with any of the

plasmids in NIH 3T3 or MDCK cells (Fig 4) Only

slight activation with the constructs p-187 and p-126

was observed in CaCo2 cells Thus, the full length

(p-1453) as well as the minimal (p-126) promoter

constructs were able to mediate liver restricted luciferase reporter expression

Effect of bile acids on Bsep promotor function

in HepG2 cells Bile acid dependancy of rat Bsep promoter activity was analysed by transfecting the full length (p-1453) and the minimal (p-126) promotor constructs into HepG2 cells in the presence of various bile acids (Fig 5) Expressed luciferase activities were higher with the minimal p-126 (Fig 5B) as compared with the full length p-1453 (Fig 5A) promoter construct While most of the uncon-jugated bile acids exerted positive effects on Bsep reporter activity, the taurine conjugated bile salts tauro-cholic acid and tauroursotauro-cholic acid (TUDCA) had virtually no effect The strongest reporter gene activation occurred with the primary bile acid chenodeoxycholic acid (CDCA) in p-126 transfected cells (230 ± 18% of controls) This CDCA-mediated stimulation of luciferase activities was concentration dependent for both reporter

Fig 2 Nucleotide sequence of the 5¢ flanking region of the rat Bsep gene A total of 2488 bp has been sequenced For better understanding only part of this sequence is presented Nucleotides are numbered relative to the major transcription initiation site (nucleotide +1; arrow) Potential binding sites for cis-acting elements are underlined with their names indicated above The TATA motif is boxed The minor transcription start site at nucleotide +10 is marked by an asterisk The complete nucleotide sequence of the Bsep promoter is available in the GenBank and EMBL databases under the accession number AF452071.

gene constructs Weaker activation of the Bsep promoter

was observed with the secondary bile acids deoxycholic

acid (DCA) and lithocholic acid (LCA) with maximal

effects in p-126 transfected cells at 100 lM (170 ± 12%)

and 50 lM (160 ± 11%), respectively The

taurine-con-jugated dihydroxylated bile acids taurodeoxychenocholic

acid (130 ± 5%, p-126) and taurochenodeoxycholic acid

(135 ± 6%, p-126) exhibited only slight stimulations of the Bsep promoter, and the trihydroxylated conjugates taurocholic acid (98 ± 10%, p-126) and tauroursode-oxycholic acid (90 ± 12%, p-126) had no stimulatory effects at all Mutation of the FXRE-motif immediately upstream of the TATA box in the p-126 minimal promoter construct completely abolished the stimulation

of luciferase activity by CDCA in HepG2 cells (Fig 6) Interestingly CDCA reduced the activity of the mutant m-126 Luc even below that of the wild-type p-126 Luc

Analysis of the binding affinity of the putative FXRE site for the orphan nuclear receptor FXR

To characterize the FXRE site identified in the Bsep promoter EMSA were carried out using an oligonucleotide corresponding to the nucleotide sequence of the first FXRE

Fig 3 Deletional analysis of the rat Bsep promoter activity in

trans-fected HepG2 cells Varying lengths of the 5¢ region of the Bsep gene,

from )1453 to )27 bp relative to the transcription start site and

extending to nucleotide +80, were amplified by PCR using the

genomic clone as a template and then inserted into the promoterless

luciferase vector pGL3 basic The plasmid p+80–1453 containing the

same nucleotide sequence in antisense orientation served as a control.

The constructs were transiently cotransfected with a renilla luciferase

expression plasmid (pRL-TK) into HepG2 cells as described in

Materials and methods Luciferase activity of each construct was

determined as relative light units of firefly luciferase per relative light

units of renilla luciferase (luc/ren) All values were expressed relative to

the longest construct p-1453, which was assigned 100% Transfections

were carried out in triplicate, and repeated three times Data are

the means ± 1 SD.

Fig 4 Functional analysis of the Bsep promotor in cell lines The

constructs p-1453, p-187, p-126 and p+80–1453 were cotransfected

with pRL-TK into four different cell lines given on the left of the panel.

Activities are expressed as relative light units of luciferase activity per

relative light units of renilla luciferase activity Transfections were

carried out in triplicate Data are expressed as means ± SD of three

individual experiments.

Fig 5 Effect of bile acids on Bsep promotor function in HepG2 cells (A) Reporter gene activity after transfection in triplicate with p-1453, the longest construct containing all upstream regulatory elements Cells were subsequently incubated for 48 h with the indicated bile acids

at the concentrations stated in Materials and methods Promotor activities were determined relative to untreated controls which were set 100% Data are the means ± SD of at least three individual experi-ments (B) Transfection with p-126, the minimal Bsep promoter con-taining one FXRE close to the TATA motif Incubation with bile acids and assays of promotor activities were carried out as in (A).

*P < 0.05.

element immediately adjacent to the TATA box (Fig 7).

The labelled probe was incubated with nuclear extracts from

rat liver A specific slowly migrating complex was formed

(lanes 1 and 2) No complex could be detected in the

presence of excess unlabelled oligonucleotide as specific

competitor (lane 9) whereas formation of the complex was

unaffected by excess of a nonspecific competitor (lane 10)

Addition of either a specific antibody against the

nuclear receptor FXR (lanes 3 and 4), or against retinoid

X receptor (RXR) (lanes 5 and 6) or a combination of both

(lanes 7 and 8) resulted in a reduction of signal intensities of

the specific band Supershifted bands were rather weak and only detectable after overexposure of the autoradiograph These findings are consistent with the observation that for transcriptional regulation FXR together with RXR form a heterodimer that subsequently binds to FXRE elements of bile acid-sensitive genes

Regulation of Bsep promoter function by drugs The ability of drugs to regulate Bsep gene transcription was assessed by transfection of the full length construct p-1453 into HepG2 cells and subsequent treatment of these cells with various compounds The promoter activity was significantly reduced by rifampin (77 ± 7%) and b-estra-diol (77 ± 6%) (Fig 8) In contrast, the estrogen antag-onist tamoxifen induced Bsep promoter activity (134 ± 15%) No significant effect was observed following treat-ment with the steroids dexamethasone (98 ± 9%) and hydrocortisone (91 ± 13%) or with the narcotic pheno-barbital (111 ± 24%)

D I S C U S S I O N

Understanding mechanisms which preserve continuous bile flow under physiologic and cholestatic conditions requires knowledge of Bsep promoter function Recently a sophis-ticated network has been revealed comprised of orphan nuclear receptors as feed-back regulators for the synthesis, hepatocellular uptake and excretion of bile acids [6,7,13–15] Furthermore transcriptional regulation of Bsep might play a critical role in drug-induced cholestasis Thus, we present the nucleotide sequence, as well as a systematic structural and functional analysis of the 5¢ flanking region of the Bsep gene

The 5¢ deletional analysis of the Bsep promoter revealed a region from nucleotide)126 to nucleotide )27 providing basal activity at similar levels as compared with the longest reporter construct extending to nucleotide)1453 upstream from the transcription start site The former region was

Fig 7 Binding activity of hepatic nuclear extracts to an oligonucleotide

containing the FXRE Hepatic nuclear extracts were prepared from

untreated rats Nuclear extracts (5–10 lg protein) were incubated with

a radiolabelled oligonucleotide containing the FXRE binding site,

electrophoresed through a 6% nondenaturing polyacrylamide gel and

autoradiographed For supershift experiments nuclear extracts were

preincubated with polyclonal antibodies against either FXRa (lane 3

and 4), RXRa (lane 5 and 6) or both (lane 7 and 8) prior to incubation

with the specific oligonucleotides Samples represented in lanes 9 and

10 were incubated in the presence of unlabelled specific (SC) and

nonspecific (NSC) competitor DNA at 100-fold molar excess.

Fig 6 Decrease of basal activity and loss of CDCA-mediated

stimu-lation of the minimal Bsep promoter after mutation of the FXRE The

wild-type (p-126) or the mutated (m-126) minimal Bsep reporter

plasmid were transfected into HepG2 cells, as described in Fig 5.

Luciferase activities were determined following a 48 h incubation in

the presence or absence of 100 l M CDCA *P < 0.05.

Fig 8 Effects of endogenous substrates and drugs on Bsep promoter function The full length Bsep reporter plasmid (p-1453) was trans-fected into HepG2 cells and luciferase activities were measured after a

48 h incubation period in the presence of the indicated compounds The following substrate concentrations were used: 5 lmolÆL)1each of dexamethasone, hydrocortisone, and b-estradiol; 50 lmolÆL)1each of phenobarbital, rifampin, and tamoxifen Assay conditions were the same as described in Fig 5 *P < 0.05.

therefore designated the minimal Bsep promoter,

appar-ently containing all binding sites required for basal

transcription The minimal promoter and larger constructs

were only functional in the human hepatoblastoma derived

cell line HepG2, indicating a liver-specific activity

Trans-acting factors directing liver-specific gene expression include

a set of liver-enriched factors, such as HNF1, HNF3b,

HNF4, and C/EBPb [16,17] Among these factors only a

putative binding site for HNF3-b overlapping with the

TATA box of transcription initiation could be found within

the minimal Bsep promoter Additional consensus motifs of

the minimal promoter include three AP1 sites and a

CCAAT box As opposed to the apparent importance of

HNF1a in liver-specific expression of the basolateral

transporters Ntcp and organic anion transporting

polypep-tide (OATP)-C [11,18] and other genes, including

cyto-chromes P450 [19], albumin and a1-antitrypsin [20] a

consensus motif for HNF1a could not be detected in the

minimal rat Bsep promoter Basal transcription of the major

canalicular organic anion exporter Mrp2, another member

of hepatocellular ABC transporters, was also not dependent

on HNF1 [21] Thus, minimal promoter activity and

liver-specific transcription of canalicular ABC transporters

appear not to require HNF1 but seem to be controlled by

other factors

Further upstream of the minimal promoter several

putative binding sites for liver enriched and ubiquitously

expressed transcription factors were located The function

of the general DNA elements, including sites for the

octamer binding proteins, NF1 and SP1 need further

evaluation Of note were five HNF3-b motifs in close

vicinity between nucleotide )718 and nucleotide )550

preceeded by a HNF1a site In this study liver specificity

of Bsep transcription was not dependent upon the combined

action of HNF1 and HNF3 as was reported for the human

glucose transporter type 2 isoform gene [22] Therefore the

close distribution of these sites seems to play a different role,

e.g in developmental regulation of Bsep expression

Interestingly, the 5¢ deletional analysis resulted in

repor-ter gene activities that were distributed in a bimodal manner

(Fig 3) Peak luciferase activities were obtained with the

constructs extending to nucleotide )1453 and nucleotide

)187, respectively, while transfection of the p-800 plasmid

resulted in < 10% relative promoter activity but rapidly

increased with further progressive deletions This suggests

the influence of a strong inhibitory cis-acting element

located between nucleotide )800 and nucleotide )512

Among known inhibitory consensus motifs, including AP-2,

PuF, CREBand Ets, only a MyoD site was detected

between nucleotide)797 and nucleotide )787 that could

potentially mediate a repressive effect on Bsep gene

transcription Apart from its function as an activator in

skeletal muscle differentiation [23] MyoD has been

des-cribed as an inhibitor of cell proliferation [24] and repressor

of the myogenic HLH Myf-5 gene expression [25]

Bile secretion and enterohepatic circulation of bile acids

are critically dependent on two key transport systems of

hepatocytes: The Ntcp (sodium-taurocholate

cotransport-ing polypeptide) mediates basolateral uptake of bile acids

whereas Bsep mediates their excretion into the biliary tract

There is good evidence from studies on animal models that

these transporters are inversely regulated in response to

cholestatic conditions and during liver regeneration [3,4]

with a pattern of diminished Ntcp expression and sustained

or even up-regulated Bsep expression The purpose of this regulatory scheme is to protect hepatocytes against accu-mulation of toxic bile acids by preventing their further uptake and preserving or enhancing their excretion The discovery of bile acids as ligands of the orphan liver receptor FXR [14] led to the concept of FXR-mediated regulation of Ntcp and Bsep Indeed, studies in mice, deficient for FXR clearly demonstrated a failure to appropriately adjust Ntcp and Bsep transcription after feeding with cholic acid [5] and thus these mice rapidly developed liver injury While Ntcp down-regulation by bile acid-bound FXR was mediated indirectly requiring the additional action of a small heterodimeric partner [7], binding of FXR to an IR-1 element within the 5¢ flanking region of the human BSEP gene was recently confirmed [6] In the present study we found a similar IR-1 element at an identical position (nucleotide)52 to nucleotide ) 64) in the rat Bsep promoter and demonstrated binding of the RXRa/FXR heterodimer

to this element in an electrophoretic retardation assay In both species the IR-1 motifs were adjacent to or in the case

of the rat even overlapping with a TATA consensus sequence Furthermore a binding site for HNF3-b or HNF-5 was also located immediately nearby in the rat and human Bsep promoter, respectively The conserved local-ization within a similar surrounding indicates the functional importance of the IR-1 element in mammalian Bsep/BSEP expression and suggests its interaction with members of the HNF transcription factor family and general factors of the transcription initiation complex In addition, the IR-1 element obviously contributes to basal activity of the Bsep minimal promoter, because mutagenesis of this site signifi-cantly reduces the relative reporter gene activity to 80% of wild-type controls (Fig 6) The intrinsic bile acid synthesis

in HepG2 cells [26], namely of CDCA and CA further supports this concept The full length Bsep promoter was stimulated by a number of conjugated and unconjugated bile acids in HepG2 cells The primary bile acid CDCA was the most potent stimulator, followed by DCA and LCA As expected from the localization of the IR-1 element stimu-lation of reporter gene activity could also be observed with the minimal promoter extending 126 bp upstream of the transcription initiation site Using this construct stimulation

of luciferase activities by the unconjugated bile acids CDCA and DCA was clearly concentration dependent In contrast treatment with LCA, a hydrophobic secondary bile acid resulted in lower promoter activities above 50 lM, due to its known cytotoxic effects [27] Mutation of the IR-1 element within the minimal promoter resulted in a loss of CDCA reporter stimulation, indicating the functional importance

of this binding site for bile acid-mediated Bsep regulation Although conjugated bile acids are the predominant form found in bile [28] they were either weak stimulators of the Bsep minimal promoter (taurodeoxychenocholic acid) or even failed to enhance reporter activity (TC, TUDCA) Previous studies showed that conjugated bile acids could only activate FXR after they have been transported into the cells by a membrane carrier [14] As HepG2 cells express the human liver organic anion transporting polypeptide OATP-A (previously called OATP) in their plasma mem-brane they are capable of taking up taurin-conjugated bile acids [29] that can bind to FXR The weak Bsep promoter stimulation by conjugated bile acids could be explained by

lower intracellular concentrations compared with their

conjugated forms, despite the carrier-mediated uptake

Drug-induced cholestasis is a common phenomenon in

medical treatment The molecular mechanisms of

drug-induced liver injury include impairment of hepatocellular

bile secretion, obstructive cholangiolitis and sclerosing

cholangitis [30] Bile secretion is dependent on the amount

of Bsep expressed in hepatocellular canalicular membranes

As substrates, such as b-estradiol and rifampin inhibit Bsep

promoter activity they are capable of causing cholestasis by

the reduction of canalicular bile acid transport capacity In

contrast with b-estradiol the anti-estrogen tamoxifen

stimu-lated Bsep promoter activity (Fig 7) Estrogen binds to

intracellular receptor proteins that subsequently act on

target genes either by binding to-specific estrogen response

elements or by stimulating the activity of factors of the AP1

complex [31,32] Both elements are located on the Bsep

promoter and could therefore potentially be involved in

estrogen-mediated modification of Bsep gene transcription

In analogy to the human choline acetyltransferase and the

lipoprotein lipase gene the inhibition of the Bsep promoter

activity by estrogens may also be mediated by AP1 or

AP1-like recognition sites [33,34]

In summary, we have identified the rat Bsep gene

promoter and characterized its activity in different cell lines

Bsep promoter function was restricted to

hepatocyte-derived HepG2 cells Comparative sequence analysis of

the 5¢ flanking region of the rat Bsep gene revealed binding

sites for liver enriched and ubiquitously expressed

tran-scription factors We have located FXRE consensus sites

and demonstrated the functional importance of the FXRE

immediately upstream of the TATA box in bile acid

negative feed-back regulation There is evidence for

differ-ential modulation of Bsep gene transcription by various

compounds Bsep promoter analysis in the presence of

drugs could serve as a useful tool in predicting drug-induced

cholestasis caused by impaired gene transcription

A C K N O W L E D G E M E N T S

Supported in part by grants from the Deutsche

Forschungsgemein-schaft to T.G (Ge 812/2-1) and C.G (SFB542, Teilprojekt C1).

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