Báo cáo Y học: Functional analysis of the rat bile salt export pump gene promoter Regulation by bile acids, drugs and endogenous compounds potx

Functional analysis of the rat bile salt export pump gene promoter Regulation by bile acids, drugs and endogenous compounds Thomas Gerloff 1 , Andreas Geier 2 , Ivar Roots 1 , Peter J. Meier 3 and Carsten Gartung 2 1 Institute of Clinical Pharmacology, Charite ´ University Medical Center, Humboldt University, Berlin, Germany; 2 Department of Internal Medicine, Aachen University of Technology, Aachen, Germany; 3 Division 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) Eur. J. Biochem. 269, 3495–3503 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03030.x 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. MATERIALS AND METHODS 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 database TRANSFAC 4.0 (MatInspector V2.2, http:// transfac.gbf.de). Construction of plasmids Aseriesof5¢ 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 MluIandBglII. 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 Quickchange TM 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 37 °C24hpriorto transient transfections. Using Tfx TM -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 lgtotalRNA 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 nucleotide 62 of the Bsep cDNA was radiolabeled at the 5¢endwith[c- 32 P]ATP (Ambion, AMS Biotechnology, Germany) to perform an S1-nuclease assay for the exact localization of the transcrip- tion initiation site. Total RNA from rat liver (100 lg) was hybridized with the labelled antisense probe at 42 °Cfor2 h (Ambion, Germany) and subsequently digested with S1 nuclease for 30 min at 37 °C. 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 32 P-end-labeled oligonucleotide probe (2 · 10 4 c.p.m.) in a 20-lL reaction containing 8 lLwater, 4 lL5· binding buffer (25 m M Hepes pH 7.6, 50 m M KCl, 0.5 m M dithiothreitol, 5 m M MgCl 2 ,0.5m M EDTA, 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 3496 T. Gerloff et al. (Eur. J. Biochem. 269) Ó FEBS 2002 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. RESULTS Determination of the transcriptional start site of the Bsep 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 the Bsep 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 V 2.1). 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. Ó FEBS 2002 Rat Bsep promoter analysis (Eur. J. Biochem. 269) 3497 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 tauroursocholic 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. 3498 T. Gerloff et al. (Eur. J. Biochem. 269) Ó FEBS 2002 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 l M (170 ± 12%) and 50 l M (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. Ó FEBS 2002 Rat Bsep promoter analysis (Eur. J. Biochem. 269) 3499 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%). DISCUSSION 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 )1 each of dexamethasone, hydrocortisone, and b-estradiol; 50 lmolÆL )1 each of phenobarbital, rifampin, and tamoxifen. Assay conditions were the same as described in Fig. 5. *P < 0.05. 3500 T. Gerloff et al. (Eur. J. Biochem. 269) Ó FEBS 2002 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 a 1 -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, CREB and 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 l M , 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 Ó FEBS 2002 Rat Bsep promoter analysis (Eur. J. Biochem. 269) 3501 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. ACKNOWLEDGEMENTS Supported in part by grants from the Deutsche Forschungsgemein- schaft to T.G. (Ge 812/2-1) and C.G. (SFB 542, Teilprojekt C1). REFERENCES 1. Gerloff, T., Stieger, B., Hagenbuch, B., Madon, J., Landmann, L., Roth, J., Hofmann, A.F. & Meier, P.J. 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Biochem. 269) 3503 . Functional analysis of the rat bile salt export pump gene promoter Regulation by bile acids, drugs and endogenous compounds Thomas Gerloff 1 , Andreas. studied yet. To analyse systematically the rat Bsep promoter and to investigate the regulation of Bsep transcription by bile acids, drugs and endogenous compounds