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Am J Physiol Gastrointest Liver Physiol 289: G831-G841, 2005. First published April 28, 2005; doi:10.1152/ajpgi.00307.2004
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INFLAMMATION/IMMUNITY/MEDIATORS

Cytokine-dependent regulation of hepatic organic anion transporter gene transactivators in mouse liver

Andreas Geier,1 Christoph G. Dietrich,1 Sebastian Voigt,1 Meenakshisundaram Ananthanarayanan,2 Frank Lammert,1 Anne Schmitz,1 Michael Trauner,3 Hermann E. Wasmuth,1 Diana Boraschi,4 Natarajan Balasubramaniyan,2 Frederick J. Suchy,2 Siegfried Matern,1 and Carsten Gartung1

1Department of Internal Medicine III, Aachen University, University Hospital, Aachen, Germany; 2Department of Pediatrics, Mount Sinai Medical Center, New York, New York; 3Division of Gastroenterology and Hepatology, Department of Internal Medicine, Medical University Graz, Graz, Austria; and 4Institute of Biomedical Technologies, University of Pisa, Consiglio Nazionale delle Ricerche, Pisa, Italy

Submitted 13 July 2004 ; accepted in final form 26 April 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Proinflammatory cytokines such as TNF-{alpha} and IL-1{beta} lead to downregulation of hepatic organic anion transporters in cholestasis. This adapted response is transcriptionally mediated by nuclear hormone receptors and liver-specific transcription factors. Because little is known in vivo about cytokine-dependent regulatory events, mice were treated with either TNF-{alpha} or IL-1{beta} for up to 16 h. Transporter mRNA expression was determined by Northern blot analysis, nuclear activity, and protein-expression of transactivators by EMSA and Western blotting. TNF-{alpha} induces a sustained decrease in Ntcp, Oatp1/Oatp1a1, and Bsep mRNA expression but exerts only transient [multidrug resistance-associated protein 2 (Mrp2)] or no effects (Mrp3) on Mrps. In addition to Ntcp and Oatp1/Oatp1a1, IL-1{beta} also downregulates Bsep, Mrp2, and Mrp3 mRNAs to some extent. To study transcriptional regulation, Ntcp and Bsep promoters were first cloned from mice revealing a new distal Ntcp hepatocyte nuclear factor 1 (HNF-1) element but otherwise show a conserved localization to known rat regulatory elements. Changes in transporter-expression are preceeded by a reduction in binding activities at IR-1, ER-8, DR-5, and HNF-1{alpha} sites after 4 h by either cytokine, which remained more sustained by TNF-{alpha} in the case of nuclear receptors. Nuclear protein levels of retinoid X receptor (RXR)-{alpha} are significantly decreased by TNF-{alpha} but only transiently affected by IL-1{beta}. Minor reductions of retinoic acid receptor, farnesoid X receptor, pregnane X receptor, and constitutive androstane receptor nuclear proteins are restricted to 4 h after cytokine application and paralleled by a decrease in mRNA levels. Basolateral and canalicular transporter systems are downregulated by both cytokines, TNF-{alpha} and IL-1{beta}. Activity of HNF-1{alpha} as regulator of mNtcp is suppressed by both cytokines. Decreased binding activities of nuclear receptor heterodimers may be explained by a reduction of the ubiquitous heterodimerization partner RXR-{alpha}.

cholestasis; tumor necrosis factor-{alpha}; interleukin-1{beta}; nuclear hormone receptors; gene expression

SYSTEMIC OR LOCAL INFLAMMATORY conditions such as bacterial infection or hepatitis are frequently associated with cholestasis (43). In animal models of inflammation, administration of endotoxin or single cytokines leads to a markedly impaired bile flow (7, 44, 47). This reduction in bile flow has been attributed to an inflammation-mediated downregulation of hepatic organic anion transporters (37, 52). A number of class II nuclear hormone receptors and liver enriched transcription factors have been identified to mediate this complex and coordinated downregulation (30, 50). The effects of endotoxin treatment on transporter expression (23, 27, 36, 40, 42, 51) and the activity of corresponding transcription factors (50) have been studied in detail. Endotoxin treatment leads to downregulation of sodium-dependent taurocholate cotransporter Ntcp (Slc10a1) protein expression, mRNA levels, transcription initiation, and reduced nuclear levels of its key transactivators hepatocyte nuclear factor 1{alpha} (HNF-1{alpha}; TCF1) and the nuclear receptor heterodimer retinoid X receptor {alpha}:retinoic acid receptor {alpha} (RXR{alpha}:RAR{alpha}) (50). Subsequently, downregulation of other hepatic transporter systems, such as the multidrug resistance-associated protein 2 Mrp2 (Abcc2) (33, 48, 51) and the bile salt export pump Bsep (Abcb11) (40), has been observed at both protein and mRNA levels in response to endotoxin.

Much less is known about the particular contribution of proinflammatory cytokines in vivo that mediate many of the effects of endotoxin (14). TNF-{alpha} and IL-1{beta} have been shown to downregulate mRNA levels of Ntcp (23), organic anion transporting polypeptides Oatp1/Oatp1a1 (Slco1a1), Oatp2/Oatp1a4 (Slco1a4), Mrp2 (Abcc2), Mrp3 (Abcc3), and Bsep in mice (27). However, mRNA expression data were obtained from different mouse strains with conflicting results. In addition, the molecular mechanisms by which these cytokines regulate transporter gene transcription have only been studied in cell culture systems (11, 41), with the exception of the RXR NR2b1, which is at least decreased at the mRNA level in hamsters by both TNF-{alpha} and IL-1{beta} (4). Because class II nuclear hormone receptors are also key regulators in cholesterol and lipid metabolism (10, 46), regulatory events under inflammatory conditions in vivo are not restricted to biliary transport alone but are of general biological importance in regard to hepatic metabolism. Thus, in the present study, we determined the particular contribution of TNF-{alpha} and IL-1{beta} in an orchestrated regulation of a variety of hepatobiliary transporter genes and their corresponding transcription factors including nuclear availability and binding activity in C57BL/6 mice.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal models. Eight- to twelve-week-old male C57BL/6NTac mice (20–25 g) were obtained from Taconic M&B (Ry, Denmark) and bred under specific pathogen-free conditions with a 12:12-h light-dark cycle. Livers were harvested from nonfasted mice 4, 8, and 16 h after intraperitoneal injection of either 6 µg of recombinant human TNF-{alpha}, 1 µg of recombinant human IL-1{beta}, or saline (control) according to Green et al. (23) as well as untreated animals. The livers were immediately frozen in liquid nitrogen. All study protocols were approved by the Federal Government’s Animal Care Committee.

Northern blot analysis. RNA was isolated from the liver by standard phenol chloroform extraction procedure. Total RNA (10–20 µg) was analyzed by Northern blot analysis as previously described (20). Mouse-specific cDNA probes for Oatp1/Oatp1a1 (Slco1a1), Mrp2, Mrp3, Bsep, and Gapdh were cloned from the corresponding cDNAs after RT-PCR or derived from expressed sequence tag clones with >95% homology to the previously identified genes, as previously described (15). In the case of Ntcp, a cross-reacting rat cDNA was employed (23).

Cloning of mouse Ntcp promoter. A mouse 129SVJ genomic library (Stratagene) constructed in the LambdaFixII vector was screened with a partial mouse Ntcp cDNA fragment using standard screening protocols (2). DNA from one lambda clone after verification by Southern blot analysis and restriction enzyme digestion was sequenced using a primer derived from a 5'-region of mouse Ntcp cDNA. With the use of this sequence information (GenBank accession number AF190698), 1.1- and 0.6-kb fragments were amplified by PCR with Pfu polymerase, digested with KpnI and BglII, and cloned into pPX2{Delta}2 (25). The HNF1 site at –646/–630 in the mouse Ntcp promoter was mutated using the following primers: forward, 5'-cctcaggcaccagctgtccacgcttcacagctggag-3'; and reverse, 5'-caccagctgtgaagcgtggacagctggtgcctgagg-3' (mutated bases indicated in bold). Quicksite site-directed mutagenesis kit (Stratagene, La Jolla, CA) was employed to create the mutations according to the manufacturer’s instructions. Mutated clones were verified by automated fluorescent sequencing for the authenticity of the mutations.

Cloning of mouse Bsep promoter. The sequence of the 5'-flanking region was generated up to –1785 bp by adapter-ligated PCR (AF303740 [GenBank] ) (49), employing the Universal Genome Walker Kit (Clontech), and by primer walking on bacterial artificial chromosomes (BACs) following standard protocols (5). BACs containing the whole Bsep gene were identified by screening a genomic BAC library (RPCI-23) from inbred mouse strain C57BL/6J (BACPAC Resources, Roswell Park Cancer Institute, Buffalo, NY), as described previously (17). The minimal promoter –376/+64 bp was amplified by PCR with Pfu polymerase and inserted into the multiple cloning site of pGL3 basic (Promega).

Analysis of 5'-untranslated regions of mouse transporter genes. The 5'-untranslated regions of the mouse genes were analyzed in the promoter sequence of Ntcp (AF190698 [GenBank] ) and Bsep (AF303740 [GenBank] ) and compared with the rat sequence of respective genes (21, 31). The mouse Mrp2 5'-untranslated region was localized by alignment of the rat promoter sequence (Y14995 [GenBank] ) (33) upstream to the Mrp2 locus in the recently published mouse genome sequence (24).

Cell culture and transient transfections. HepG2 hepatoma cells (1 x 105 cells/well) were grown in MEM or DMEM/F-12 (GIBCOBRL) supplemented with 10% fetal calf serum and penicillin/streptomycin. Transfections of Ntcp promoter constructs were performed utilizing Fugene6 (Roche, Indianapolis, IN) according to manufacturer’s instructions. In cotransfection experiments, 1 µg of –1.1-kb mNtcp (wild type or HNF-1 mutant) or –0.6 mNtcp pXP2{Delta}2 reporter was combined with 0.2 µg of HNF-1{alpha} or HNF-1{beta} expression plasmids (obtained from Dr. F. J. Gonzalez, National Cancer Institute/National Institutes of Health, Bethesda, MD; and Dr. G. R. Crabtree, Stanford University). Cointroduced pCMV{beta}-Gal expression vector served as an internal control for transfection efficiency.

For the analysis of Bsep promoter constructs, HepG2 cells were transfected by calcium phosphate coprecipitation with 2 µg of –376-bp mBsep constructs per well together with 0.037 µg pRL-TK (Promega), permitting normalization for transfection efficiency. In addition, cotransfections were performed by adding 150 ng of human retinoid X receptor (FXR) and RXR expression plasmids (obtained from Dr. David Mangelsdorf; University of Texas Southwestern, Dallas, TX) in the presence or absence of chenodeoxycholic acid (100 µM). In a subset of experiments, recombinant TNF-{alpha} (60 ng/ml) or IL-1{beta} (10 ng/ml) was added to the medium. Both luciferase assays were performed according to the manufacturer’s instructions (Promega).

Electrophoretic mobility shift analysis. Preparation of nuclear extracts and electrophoretic mobility shift assays were performed as previously described (20). Protein (5–10 µg) was incubated on ice for 30 min with 2 x 104 cpm 32P-end-labeled oligonucleotide probes. For competition assays, 100-fold molar excess of unlabeled oligonucleotides were coincubated with the labeled probe. For supershift experiments, nuclear extracts were preincubated for 30 min on ice with 1 µg of a polyclonal antibody against HNF-1{alpha} or FXR (Santa Cruz Biotechnology) before addition of labeled oligonucleotides. Separation of protein-DNA complexes was performed by electrophoresis through a nondenaturing 6% polyacrylamide gel and quantified by phosphoimaging.

Western blot analysis. Preparation of either liver microsomes or nuclear extracts was performed as previously described (20) to analyze protein levels of Ntcp as well as the transcription factors RXR{alpha}, RAR{alpha} (NR1b1), FXR (NR1h4), pregnane X receptor (PXR; NR1i2), constitutive androstane receptor (CAR; NR1i3), and HNF-1{alpha}. Nuclear protein concentrations were determined according to Bradford (8). Microsomal (75 µg) or nuclear (10–25 µg) protein extract was separated by SDS-PAGE (38) and probed to anti-rat Ntcp fusion protein antiserum (20), monoclonal anti-Na+-K+-ATPase-{alpha}1 antibody (Upstate), or respective transcription factor antibodies (Santa Cruz Biotechnology) after transfer to blotting membranes. Immune complexes were detected according to the ECL kit (Amersham).

RT and PCR. RT and subsequent TaqMan real-time PCR on an ABI Prism 7900HT sequence detection system (Applied Biosystems, Foster City, CA) was performed as previously described (16). The following primers and flourogenic probes were used: FXR: forward, 5'-CGGCTGTCAGGATTTGTGC-3'; reverse, 5'-GTTGTATGGGGAGTACGATTC-3'; and probe, 5'-FAM-CCAGCTAAAGGTATGCTAACAGAACACGC-TAMRA-3', corresponding to GenBank accession number NM_009108; PXR: forward, 5'-CCTTTGACACAACTTTCTCCCAC-3'; reverse, 5'-CAGGGTCTTCCAACAGTGAG-3'; and probe, 5'-FAM-CAAGGATTTCCGGCTGCCTGCAGTGTTCC-TAMRA-3', corresponding to GenBank accession number NM_010936; and CAR: forward, 5'-CCACAGGCTATCATTTCCACG-3'; reverse, 5'-CAAACGGACAGATGGGACCA-3'; and probe, 5'-FAM- CTGCAAGGGCTTCTTCAGACGAACAGTCAGC-TAMRA-3', corresponding to GenBank accession number NM_009803. All data were normalized to 28S rRNA (52). RT and competetive PCR for RAR{alpha}, and RXR{alpha} was performed as previously described in detail (15). The following primers and mutagenic primers as internal standards were used: RAR{alpha}: forward, 5'-AGAGCAGCAGTTCCGAAGAG-3'; reverse, 5'-AGCCTTGAGGAGGTTGATCT-3'; and mutagenic primer, 5'-CGGATCCCCTTGTTGATGAT-3', corresponding to GenBank accession number NM_009024 and using BamH1 as restriction enzyme; and RXR{alpha}: forward, 5'-CTCCTATCAGCACCCTGAGC-3'; reverse, 5'-GTCTCAGTCTTGGGCTCGAC-3'; and mutagenic primer, 5'-CTTGTGGATCCGGCAGTCCT-3', corresponding to GenBank accession number NM_011305 and using BamH1 as restriction enzyme. Annealing temperature for RAR{alpha} and RXR{alpha} was 60°C.

Statistical analysis. Statistical analysis between controls and cytokine-treated animals was performed using multivariate ANOVA with posttesting. Statistical significance was assumed at P values of <0.05. Data represent means ± SD of four animals per group.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effects of inflammatory cytokines on mRNA and protein expression of hepatobiliary transporter genes. Application of TNF-{alpha} induced a sustained decrease in Ntcp, Oatp1/Oatp1a1, and Bsep mRNA expression by 70% compared with controls up to 16 h (Fig. 1). In comparison, effects on Mrp2 mRNA expression were moderate and of short duration (–50% at 4 h) or, in the case of Mrp3, not significant at all (Fig. 1). IL-1{beta} exerted similar effects as TNF-{alpha} on Ntcp and Oatp1/Oatp1a1 mRNA expression, which rapidly declined to a minimum of 50% of controls. Downregulation of Oatp1/Oatp1a1 mRNA by IL-1{beta} appeared to be of shorter duration compared with TNF-{alpha}-treated animals. In contrast to TNF-{alpha}, downregulation of the canalicular Bsep by IL-1{beta} was less pronounced and significantly reduced only at 8 h after injection by –50% of controls. For both Mrps, regulation by IL-1{beta} appears more important, because Mrp3 mRNA declined after 8 h by 50% of controls, whereas the canalicular Mrp2 was rapidly and profoundly downregulated by 70% (Fig. 1, C and D). Both TNF-{alpha} and IL-1{beta} treatment already moderately affected transporter protein expression after 16 h as shown for Ntcp (Fig. 2).



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  		Fig. 1. Effect of TNF-{alpha} and IL-1{beta} on steady-state mRNA levels of organic anion transporters. Total RNA was isolated from livers after 4, 8, and 16 h, blotted onto nylon membrane, and hybridized to specific cDNA probes for basolateral (Ntcp, Oatp1/Oatp1a1, Mrp3) and canalicular transporters (Bsep and Mrp2). Blots were then stripped and rehybridized with a Gapdh cDNA probe. Representative autoradiographs are shown for TNF-{alpha} (A) and IL-1{beta} (B) treatment. Autoradiographs were quantified by phosphoimaging, and data were expressed as percentage of controls (n = 4). Data are given as means ± SD. *P < 0.05 compared with controls (C and D). CO, controls; Sham, Sham controls.

 


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  		Fig. 2. Ntcp protein expression in response to proinflammatory cytokines in mice. Microsomes were prepared by differential centrifugation from livers after 4, 8, and 16 h. Protein mass of Ntcp and Na+-K+-ATPase (NK) was determined by Western blot analysis. A: representative immunoblot. Molecular weight (MW) markers are given in kDa. B: densitometric analysis. Data represent means ± SD of n = 4 animals per group expressed as percentage of controls. *P < 0.05 compared with controls.

 
Identification of mouse homologs to regulatory elements in rat transporter genes. After Ntcp and Bsep promoters were cloned (see MATERIALS AND METHODS), mouse-specific elements representing homologous sequences to established binding sites for HNF-1{alpha}, RXR:FXR (IR-1), RXR:PXR/CAR/FXR (ER-8), and RXR{alpha}:RAR{alpha} (DR-5) in rat transporter gene promoters for Ntcp, Bsep, and Mrp2 were identified in the 5'-untranslated regions of the corresponding mouse genes (31, 32, 26). As shown in Fig. 3, homologous sequences were identified in the 5'-untranslated regions of the mouse genes of Ntcp (AF190698 [GenBank] ) and Bsep (AF303740 [GenBank] ). The so-far-uncloned 5'-untranslated regions of the mouse Mrp2 were localized by alignment of the rat promoter sequence (Y14995 [GenBank] ) (33) to the recently published mouse genome sequence (24).



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  		Fig. 3. Analysis of 5'-untranslated regions of mouse transporter genes. 5'-Untranslated regions of the mouse genes were analyzed in the published mouse promoter sequence of Ntcp [GenBank accession number AF190698, counted from the rat transcription start site at position 976; mNtcp hepatic nuclear factor (HNF)-1 site at –630/–643 (mouse genome map at NW 000148.1 26171945–62); A] and Bsep [AF303740, mBsepIR-1 site at –48/–68 counted from the rat transcription start site (NT 039207.1 10293243–63); B] for regulatory elements by comparison with known rat regulatory elements. The mouse Mrp2 5'-untranslated region (C) was localized by alignment of the rat promoter sequence (Y14995 [GenBank] ) upstream to the Mrp2 locus in the mouse genome sequence (ER-8 at position NW 000148.1 4056539–58; DR-5 overlapping at position 4056528–44). Sequence homology between mNtcp and mOatp4/Oatp1b2 HNF1, mBsep and rBsep IR-1, mMrp2 and rMrp2 ER-8/DR-5 is depicted at the bottom. Cross mark, missing in mouse gene. Mouse genome map elements and positions accessible at http://www.ncbi.nih.gov/mapview/.

 
We identified a putative HNF-1{alpha} binding site in the mouse Ntcp promoter with an almost identical sequence compared with the known HNF-1 element in the mouse Oatp4/Oatp1b2 (Slco1b2) promoter (Fig. 3A) (28) . The HNF-1{alpha} binding site in the minimal promoter of the rat Ntcp gene (31) is absent in the mouse, but another potential binding site for HNF-1{alpha}, known as a key regulator of mouse Ntcp from knockout studies, could be identified at position –646/–630 bp in the upstream promoter region (AF190698 [GenBank] ; counted from the rat transcription start site at position 976). The Bsep IR-1 element is highly conserved in both rat and mouse gene promoters (Fig. 3B) (21). Alignment of the rat Mrp2 promoter sequence (33) to the region upstream of the Abcc2 locus on mouse chromosome 19 (18) (map element NW000148.1, positions 4056408–4056567 and 4056598–4056567) revealed two highly conserved sequences homologous to the known rat Mrp2 DR-5 and ER-8 elements (Fig. 3C) (32).

HNF-1 and FXR as transactivators of the mouse Ntcp and Bsep genes. To determine binding of HNF-1 to the putative newly identified binding site in the mouse Ntcp promoter, DNA binding studies with supershift of HNF-1{alpha} were performed (Fig. 4A). A specific band was observed after incubation of mouse liver nuclear extract, which could be shifted by the addition of a HNF-1{alpha} antibody and competed by 100-fold molar excess of the unlabeled oligonucleotide. To further characterize the functional relevance of HNF-1{alpha} to this upstream element in the mouse Ntcp gene, luciferase assays were performed. Cotransfection of HNF-1{alpha} and HNF-1{beta} expression plasmids increased the basal luciferase activity by 3.5 ± 0.5- and 1.5 ± 0.1-fold, respectively (P < 0.01 each; Fig. 4B), indicating transactivation by HNF-1{alpha} and, to a smaller extent, also by HNF-1{beta}. Deletion of this element at –646/–630 bp (p-607 mNtcp LUC) completely prevented the HNF-1{alpha} effect on promoter activity (Fig. 4C). Mutations of this respective element in the full promoter sequence resulted in a modest (–17%) but significant inhibition of luciferase activity (wild-type promoter + HNF-1{alpha} vs. HNF-1-mutant promoter + HNF-1{alpha}; P = 0.03; Fig. 4C). The reason for the modest inhibition on mutation of the HNF-1{alpha} cis element is not clear at present, but we postulate further HNF-1{alpha} binding sites in the distal promoter sequence or that HNF-1{alpha} may also participate with other elements in activation of the promoter in addition to binding to its own recognition sequence. Similarly, nuclear extract binds the mouse Bsep IR-1 element and forms two specific bands. Whereas the upper band could be completely shifted by addition of an FXR antibody, only an incomplete shift of the lower band occurred (Fig. 4D). Cotransfection of FXR and RXR{alpha} expression plasmids with and without the ligand chenodeoxycholic acid increased the basal luciferase activity to 11.2 ± 1.3% and 237.7 ± 63.7%, respectively (Fig. 4E). Transfection of mNtcp and mBsep full-length promoter constructs in the presence of either TNF-{alpha} or IL-1{beta} significantly decreased luciferase activity (Fig. 4, F and G).



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  		Fig. 4. Functional analysis of the mouse Ntcp and Bsep promoter. A and D: electrophoretic mobility shift assay representative autoradiographs. Hepatic nuclear extracts (10 µg protein) from untreated mice were incubated with radiolabeled oligonucleotides containing the newly identified potential HNF-1{alpha}-binding site (A) or the mBsep IR-1 element (D), electrophoresed through a 6% nondenaturing polyacrylamide gel; and autoradiographed. For supershift (lane 2) nuclear extracts were preincubated with a polyclonal antibody against HNF-1{alpha} or farnesoid X receptor (FXR), respectively, before incubation with the oligonucleotide. SC, specific competition; NSC, nonspecific competition. B, C, E, F, and G: reporter gene activity in HepG2 cells after cotransfection of HNF-1 and FXR/retinoid X receptor (RXR) expression plasmids. Reporter constructs –1.1 kb mNtcp (B), –0.6 kb mNtcp or mut-1.1kb mNtcp (C), and –376 bp mBsep (E) harboring the 5'-regulatory sequence of the mouse Ntcp and Bsep genes were transiently transfected into HepG2 cells along with HNF-1{alpha}, HNF-1{beta}, or FXR/RXR expression plasmids, respectively. In the case of FXR/RXR ligand activation was achieved by addition of 100 µM chenodeoxycholic acid (CDCA). Recombinant TNF-{alpha} or IL-1{beta} was added to either full-length mNtcp (F) or mBsep constructs (G). Transcriptional activities are given compared with the basal transcription activity. Data represent means ± SD. *P < 0.05 compared with basal luciferase activity. All transfections were performed in triplicate and were repeated twice in separate experiments. The sequence of both mouse Ntcp and Bsep constructs was verified by automated ABI sequencing.

 
Effects of inflammatory cytokines on binding activity at HNF-1 and nuclear hormone receptor regulatory elements. After analysis of the 5'-untranslated regions of mouse transporter genes, mechanisms of cytokine-induced downregulation of the corresponding hepatobiliary transporters at the mRNA level were further elucidated by electrophoretic mobility shift assays (Fig. 5, A-C). In addition to the newly identified HNF-1{alpha} element, we used oligonucleotides for IR-1, ER-8, and DR-5 sites derived from the mouse genome sequence (24), which highly correspond to the homologous responsive elements previously identified in rat transporter gene promoters for Bsep and Mrp2 (11, 21, 31, 32). In general, changes of transporter mRNA expression were preceeded by a reduction in the binding activity at HNF-1, IR-1, ER-8, and DR-5 oligonucleotides by either cytokine (Fig. 5, A and B). The suppressive effect of both TNF-{alpha} and IL-1{beta} on HNF-1{alpha} binding activity to its responsive element in the Ntcp promoter by 50 and 60%, respectively, was confined to the first 4 h. Binding to the FXR responsive Bsep IR-1 element exhibited the same time course as the mRNA expression of the corresponding gene with a more sustained TNF-{alpha} effect. Binding of the RXR{alpha}:RAR{alpha}-heterodimer to the DR-5 element decreased to 30–40% of controls. TNF-{alpha} effects on DR-5 binding were prolonged up to 8 h, and Mrp2 mRNA levels declined in parallel at this time point. No difference was observed in the cytokine-dependent downregulation of the binding activity at the Mrp2 ER-8 element. Both TNF-{alpha} and IL-1{beta} suppressed DNA binding up to 16 h by a maximum of 70–80% of controls (Fig. 5C).



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  		Fig. 5. Effect of TNF-{alpha} and IL-1{beta} on DNA binding activity at mouse regulatory elements. Hepatic nuclear extracts of either TNF-{alpha}- or IL-1{beta}-treated mice (n = 4) were analyzed as described. Representative autoradiograph of the binding activity at HNF-1{alpha}, IR-1 [binding site for FXR (49)], ER-8 [binding site for FXR, pregnane X receptor (PXR), constitutive androstane receptor (CAR) (37)] and DR-5 [binding site for RAR{alpha} (19)] oligonucleotides are shown for TNF-{alpha} (A) and IL-1{beta} (B) treatment. C: densitometric analysis. Autoradiographs were quantified by phosphoimaging, and data were expressed as percentage of controls (n = 4). Data are given as means ± SD. *P < 0.05 compared with controls.

 
Effects of inflammatory cytokines on nuclear protein levels of nuclear hormone receptors. Because three different nuclear hormone receptors (FXR, PXR, and CAR) can bind to the rat Mrp2 ER-8 element as heterodimers with RXR{alpha} (32), we performed Western blot analyses with nuclear protein extracts isolated at the same time points to quantify their particular contribution to ER-8 binding (Fig. 6, A and B). Both FXR and PXR nuclear protein levels decreased 4 h after TNF-{alpha} to a comparable extent by 50% of controls. CAR nuclear protein appeared to be decreased by TNF-{alpha} even by 60% of controls, but this was not statistically significant due to a higher standard deviation of controls. Changes observed after IL-1{beta} application were not significant. Nuclear protein levels of RAR{alpha} were reduced 4 h after TNF-{alpha} below 40% and paralleled DR-5 binding, whereas IL-1{beta} exerted minor effects. Compared with this moderate and short-lived downregulation, the nuclear protein expression of the heterodimerization-partner RXR{alpha} was virtually absent at 4 h after TNF-{alpha} and remained <30% of controls over the full study period. IL-1{beta} effects were limited to 4 h with a decrease of RXR{alpha} protein at 30% of controls and a rapid return to baseline (Fig. 6C). The reduction of HNF-1{alpha} protein levels in the presence of TNF-{alpha} and IL-1{beta} followed a similar time course as RXR{alpha} with a nadir at 4 h and explains the decreased binding activity at this time point.



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  		Fig. 6. Effect of TNF-{alpha} and IL-1{beta} on nuclear protein levels and mRNA expression of transcription factors. Hepatic nuclear protein (25 µg) of TNF-{alpha}- and IL-1{beta}-treated mice (n = 4) was separated by SDS-PAGE and probed to various antibodies. Representative immunoblot of nuclear extracts from TNF-{alpha} (A)- and IL-1{beta} (B)-treated mice. C: densitometric analysis. D: RT-PCR was performed to semiquantify mRNA levels of nuclear hormone receptors. mRNA data were normalized to those of 28S rRNA. Data represent means ± SD of n = 4 animals per group. *P < 0.05 compared with controls.

 
Inflammatory cytokines suppress mRNA expression of nuclear hormone receptors. To determine whether reduced nuclear protein expression of nuclear hormone receptors is caused by reductions in their mRNA levels, we performed RT-PCR analysis using RNA extracts isolated at the same time points (Fig. 6D). Both cytokines induced a rapid decrease in FXR and CAR mRNA expression at 4 h compared with controls (to 40 and 10%, respectively), whereas only TNF-{alpha} suppressed PXR mRNA expression (40% of controls; P < 0.05 each). For RAR{alpha}, there was only a trend toward a reduction of mRNA expression at 4 h without statistical significance. RXR{alpha} mRNA levels remained unaffected by TNF-{alpha} and appeared to be even timely increased by IL-1{beta} application.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Many of the effects of systemic or local inflammation are mediated by cytokines such as TNF-{alpha} and IL-1{beta} (14). To study their effects on transactivators of hepatobiliary transporter genes in mice, we first identified regulatory elements of Ntcp, Mrp2, and Bsep in this species. Administration of either cytokine resulted in the following findings: 1) basolateral Ntcp and Oatp1/Oatp1a1 are downregulated by both cytokines. However, TNF-{alpha} predominantly suppressed the canalicular Bsep as well as Mrp2, whereas IL-1{beta} mainly affected Mrp3. 2) HNF-1{alpha}, characterized as a regulator of basolateral Ntcp in mice, is suppressed by both cytokines. 3) Decreased binding activities of nuclear hormone receptors mainly occur by a reduction of the heterodimerization-partner RXR{alpha} and may involve both reduced nuclear protein availability and posttranslational regulatory events.

Current knowledge about mouse basolateral and canalicular organic anion transporter expression is limited to three studies in different strains of mice (6, 23, 27), which are known to differ significantly in constitutive expression of hepatobiliary transporters (39). Furthermore, contradictory results have been reported with regards to Mrp3, the only transporter system studied in two independent strains (6, 27). Therefore, we now examined the effects of TNF-{alpha} and IL-1{beta} in male C57BL/6NTc mice on mRNA expression of Ntcp, Oatp1/Oatp1a1, Mrp3, Mrp2, and Bsep. The mRNA expression of both basolateral uptake systems Ntcp and Oatp1/Oatp1a1 is rapidly downregulated by either cytokine (Fig. 1). Identical effects on Ntcp mRNA expression were observed by Green et al. (23) up to 16 h, but, in the case of Oatp1/Oatp1a1, Hartmann et al. (27) detected in CD-1 outbred mice 6 h after injection only an mRNA downregulation in response to IL-1{beta}. Furthermore, promoter studies on rat Ntcp demonstrated a 25–40% decrease in reporter gene activity on both TNF-{alpha} and IL-1{beta} treatment (11). Mrp3, the basolateral excretion system of bile acids, which is induced under cholestatic conditions (35, 45), is moderately decreased at later time points by IL-1{beta} but not TNF-{alpha} (Fig. 1). Regulation of Mrp3 by inflammatory cytokines appears to be highly strain specific because either TNF-{alpha}-mediated downregulation [CD-1 (27)] or upregulation [C57BL/6 mice (6)] was observed. In contrast to basolateral transporters, strain-specific differences in mRNA and protein expression are minor for canalicular export pumps (39). We found a downregulation of Bsep mRNA expression by either cytokine that is more prominent and sustained over 16 h in TNF-{alpha}-treated animals (Fig. 1). Although Hartmann et al. (27) also observed suppressive effects by both cytokines, IL-1{beta} appeared predominant under their conditions. Mrp2 expression is well comparable because both cytokines led to a decrease of mRNA expression between 4 and 8 h after injection, albeit it was more pronounced for IL-1{beta} in our study (Fig. 1). Studies investigating the rat gene also confirm a predominant suppressive effect of IL-1{beta} on both the rat Mrp2 promoter in HepG2 cells and rat Mrp2 gene mRNA expression in endotoxin-treated or bile duct-ligated rats (11–13, 19).

To further identify the principles of cytokine-mediated transporter gene regulation in mice, binding sites for HNF-1{alpha}, RXR:FXR (IR-1), RXR:PXR/CAR/FXR (ER-8), and RXR{alpha}:RAR{alpha} (DR-5) in mouse Ntcp, Bsep, and Mrp2 promoters were identified in the 5'-untranslated regions (Fig. 3). Our data characterize HNF-1{alpha} as a transactivator of the mouse Ntcp gene (Fig. 4, B and C), as expected from previous knockout studies (48). The newly identified HNF-1{alpha} binding site upstream of the minimal promoter region explains its transactivation activity even in the absence of the proximal HNF-1 element identified in the rat promoter sequence (31). However, a largely maintained HNF-1{alpha} transactivation despite point mutation of this respective element is strongly suggestive of further HNF-1{alpha} binding sites in the distal promoter sequence. Indeed, on the basis of a TRANSFAC sequence analysis, several additional potential HNF-1{alpha} sites (–881/–867, –810/–794, –787/–771, –751/–735, –703/–687) can be identified in the respective region of the Ntcp promoter. Regulation of the murine Ntcp promoter by HNF-1{alpha} was recently questioned by Jung and co-workers (29), who did not identify respective response elements in a 0.2-kb minimal mouse Ntcp promoter. Of interest, HNF-1{beta} also transactivates the mouse Ntcp promoter, albeit to a lesser extent. Similar to the human and rat homologs (1, 21), FXR could now be established as a ligand-activated regulator of mouse Bsep as well (Fig. 4E).

On the basis of the identification of these elements in the 5'-untranslated regions, we performed electrophoretic mobility shift assays to elucidate the mechanisms of cytokine-induced downregulation of hepatobiliary transporter genes at the mRNA level, which have not been investigated in this context to date. In parallel to a moderately reduced mNtcp promoter activity in response to proinflammatory cytokines in vitro (Fig. 4F), binding activity to the HNF-1-responsive element of the mouse promoter (Figs. 3A and 4) as well as its nuclear protein levels in vivo is reduced by at least 50% during the first 4 h after TNF-{alpha} and IL-1{beta} administration (Figs. 5 and 6). Our results are consistent with previous observations of decreased HNF-1{alpha} mRNA expression during the acute-phase response in female BALB/c mice with a 15% body surface area burn (9).

Several studies have shown that the expression and activity of various nuclear hormone receptors are rapidly decreased after endotoxin application in hamsters and C57BL/6 mice (3, 4, 33). However, the particular role of TNF-{alpha} and IL-1{beta} has only been elucidated in vivo for hamster RXR at the mRNA level (4). We therefore determined binding activity of nuclear hormone receptors to IR-1 (RXR:FXR), ER-8 (RXR:FXR/PXR/CAR), and DR-5 (RXR{alpha}:RAR{alpha}) elements identified in the mouse genes (24) (Fig. 3, B and C) as well as the nuclear protein levels for RXR{alpha} and its heterodimerization partners capable of binding the respective elements in the rat.

Binding of the DR-5 element homologous to the rat Mrp2 DR-5 RXR{alpha}:RAR{alpha}-responsive element is substantially decreased by both cytokines at 4 h with a more sustained TNF-{alpha} effect up to 8 h (Fig. 5). RAR{alpha} nuclear levels are only significantly decreased early after TNF-{alpha} application without significant changes in mRNA expression and therefore highlight the possibility of a predominant posttranslational regulation (Fig. 6). Similar findings have been reported in cell culture experiments with HepG2 cells and primary rat hepatocytes where IL-1{beta} leads to a reduced RXR{alpha}:RAR{alpha} nuclear binding by a JNK-dependent RXR phosphorylation (41). Binding to the Mrp2 ER-8 element is also suppressed by both cytokines (Fig. 5) and correlates only partially with the more IL-1{beta}-dependent downregulation of the gene (Fig. 1). We differentiated the nuclear levels and mRNA expression of the three different nuclear receptors FXR, PXR, and CAR, which bind to the rat Mrp2 ER-8 element (32) to quantify their particular contribution to ER-8 binding activity (Fig. 6). Nuclear protein levels and mRNA expression of all receptors are decreased to comparable extents after TNF-{alpha} application. In comparison, IL-1{beta}-induced signals are shortlived and lead to a modest and nonsignificant reduction of nuclear receptor protein levels. Effects of IL-1{beta} are more prominent on FXR and CAR mRNA levels and exceed the extent of protein reduction. Our results are in accordance with previous reports of PXR and CAR mRNA reduction in endotoxin-treated C57BL/6 mice (3) and a decreased TNF-{alpha}-mediated inhibition of CAR nuclear binding activity in TNF receptor p55–/–/p75–/– double knockout mice on the C57BL/6 background (53). However, these transient changes of nuclear protein levels do not explain the decreased binding-activity at the ER-8 element up to 16 h in the cases of TNF-{alpha} and IL-1{beta}. Thus other regulatory events such as phosphorylation might add to the observed effects.

The predominant TNF-{alpha} effect on RXR:FXR binding to the Bsep IR-1 element is sustained over the full study period, whereas the IL-1{beta}-mediated decrease is of limited duration (Fig. 5). Transfection studies with TNF-{alpha} and IL-1{beta} demonstrate similar effects on mBsep promoter activity (Fig. 4G). Previous transfection experiments in Hep3B cells also show an inhibition of FXR responsive element (IR-1)-luciferase activity by both inflammatory cytokines (34). Similar results were obtained in HepG2 cells with rat Bsep promoter constructs (21), where both TNF-{alpha} and IL-1{beta} suppress reporter activity (T. Gerloff and A. Geier, unpublished data). Similarly, FXR has also been shown to be decreased at the mRNA level in endotoxin-treated C57BL/6 mice (34). Although the effect of IL-1{beta} on FXR downregulation in vivo is limited in our study, the same investigators have shown a decrease of FXR mRNA by both TNF-{alpha} and IL-1{beta} at least in Hep3B cells (34).

In contrast to the moderate and timely limited downregulation of nuclear RAR, FXR, PXR, and CAR availability, the nuclear protein expression of the common heterodimerization partner RXR{alpha} is virtually absent after TNF-{alpha} and remains markedly suppressed over the full study period (Fig. 6, A and C). Therefore, decreased binding activities of nuclear hormone receptors and the coordinated downregulation of the corresponding transporter genes after TNF-{alpha} treatment mainly occur by a reduction of the heterodimerization partner RXR{alpha}. Similar findings have been observed in C57BL/6 mice on endotoxin treatment, in which RXR{alpha} is reduced in the nucleus and appears in the cytoplasm within 1 h (22). The difference between the absence of RXR{alpha} protein in the early time course and 30–40% of basal binding activity at IR-1, ER-8, and DR-5 elements renders a partial replacement of RXR{alpha}, likely by RXR{beta} or RXR{gamma}, which are almost maintained after TNF-{alpha} treatment (4). In comparison, IL-1{beta} causes only a timely limited decrease of RXR{alpha} protein (Fig. 6, B and C). Decreased binding activity at the ER-8 element, which occurs during the later time course after IL-1{beta} treatment even in the presence of sufficient nuclear receptor levels, may be explained by additional posttranslational regulatory events such as JNK-dependent RXR{alpha} inactivation via phosphorylation, which primarly occurs on IL-1{beta} signaling (41).

In conclusion, the present study illustrates in vivo the particular contribution of TNF-{alpha} and IL-1{beta} in an orchestrated regulation of hepatobiliary transporter genes and corresponding transcription factors in C57BL/6 mice. These findings expand our current knowledge of the regulatory mechanisms of a variety of hepatic acute-phase response genes and may contribute to a rational approach of a future therapy of cholestatic disorders.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This work was supported by Deutsche Forschungsgemeinschaft Grant SFB542 TP C1 (to A. Geier, S. Matern, and C. Gartung), Grant DI 729/3-1 (to C. G. Dietrich), grants from the German Network of Excellence for Viral Hepatitis (Hep-Net; to F. Lammert, H. E. Wasmuth, and S. Matern), National Institutes of Health (NIH) Grant HD-20632 (to M. Ananthanarayanan and F. J. Suchy), the Trans-NIH Mouse Initiative (to F. Lammert and A. Schmitz), and Grant P15502 [GenBank] from the Austrian Science Foundation (to M. Trauner).


   ACKNOWLEDGMENTS
 
The authors thank Aline Müller, Petra Schmitz, Dagmar Silbert, and Sonja Strauch for excellent technical assistence as well as Dr. Harald Wajant for providing recombinant TNF-{alpha}.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. Geier, Dept. of Internal Medicine III, Division of Gastroenterology and Hepatology, Aachen Univ., Pauwelsstrasse 30, D-52074 Aachen, Germany (e-mail: ageier{at}ukaachen.de)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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