Cytokine regulation of human sterol 12
-hydroxylase (CYP8B1) gene
Asmeen Jahan and
John Y. L. Chiang
Department of Biochemistry and Molecular Pathology, Northeastern Ohio Universities College of Medicine, Rootstown, Ohio
Submitted 6 May 2004
; accepted in final form 15 November 2004
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ABSTRACT
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Sterol 12
-hydroxylase (CYP8B1) catalyzes cholic acid synthesis in the liver and is feedback inhibited by bile acids. In addition to activating farnesoid X receptor (nuclear receptor subfamily 1H4), bile acids also induce inflammatory cytokines in hepatocytes. The objective of this study was to investigate the mechanism by which inflammatory cytokines inhibit human CYP8B1 gene transcription. Real-time PCR assays revealed that both chenodeoxycholic acid (CDCA) and interleukin-1
(IL-1
) markedly reduced CYP8B1, cholesterol 7
-hydroxylase CYP7A1 and hepatic nuclear factor 4
(HNF4
) mRNA expression levels in human primary hepatocytes. However, CDCA induced, but IL-1
reduced, small heterodimer partner (SHP) mRNA expression. IL-1
inhibited human CYP8B1 reporter activity only in liver cells, and a c-Jun NH2-terminal kinase (JNK)-specific inhibitor-blocked IL-1
inhibition. Activated JNK1 or c-Jun inhibited, whereas their dominant negative forms blocked, IL-1
inhibition of CYP8B1 transcription. Mutagenesis analyses mapped an IL-1
response element to a previously identified bile acid response element, which contains an HNF4
binding site. A dominant negative HNF4
inhibited CYP8B1 gene transcription and ectopically expressed HNF4
blocked IL-1
inhibition. Furthermore, IL-1
inhibited HNF4
gene transcription, protein expression, and binding to the CYP8B1 gene. JNK1 phosphorylated HNF4
and a JNK-specific inhibitor blocked the IL-1
inhibition of HNF4
expression. These results suggest that IL-1
inhibits CYP8B1 gene transcription via a mitogen-activated protein kinase/JNK pathway that inhibits HNF4
gene expression and its DNA-binding ability. This mechanism may play an important role in the adaptive response to inflammatory cytokines and in the protection of the liver during cholestasis.
bile acid synthesis; nuclear receptor; interleukin-1
; hepatic nuclear factor 4
; cholestasis
CONVERSION OF CHOLESTEROL to bile acids is the predominant pathway for catabolism of cholesterol in the body. In humans, the major bile acid biosynthetic pathway is initiated by cholesterol 7
-hydroxylase (CYP7A1) to produce two primary bile acids, cholic acid (CA) and chenodeoxycholic acid (CDCA). Sterol 12
-hydroxylase (CYP8B1) catalyzes the synthesis of CA and determines the ratio of CA to CDCA in the bile (6). Because CA is more hydrophilic than CDCA, CYP8B1 may regulate the hydrophobicity of the bile acid pool, which regulates bile acid synthesis. Bile acids excreted from the liver are stored in the gallbladder and secreted in the intestine to facilitate absorption and transport of lipid-soluble vitamins and steroids. CA is more efficient in stimulating the absorption of cholesterol in the intestine and contributes significantly to maintaining whole body cholesterol homeostasis (41). Bile acids are quantitatively reabsorbed in the ileum and transported back to the liver via the enterohepatic circulation to inhibit bile acid synthesis (6). Several experiments show that the expression of the CYP8B1 gene is repressed by bile acids, cholesterol, and insulin (5, 21, 40). The molecular mechanism of regulation of CYP8B1 by these physiological regulators remains obscure.
Targeted disruption of the Fxr gene in mice causes increased serum and hepatic bile acid, cholesterol and triglycerides, and a proatherogenic lipoprotein profile (36). In contrast to a significant increase of CYP7A1 mRNA levels in Fxr null mice, CYP8B1 mRNA expression is not altered significantly (24, 36). Bile acid feeding inhibits both CYP7A1 and CYP8B1 mRNA levels in wild-type mice but not in Fxr null mice. This is consistent with the mechanism that bile acid-activated-farnesoid X receptor (FXR) induces a negative nuclear receptor, small heterodimer partner (SHP, NR0B2), which then inhibits trans-activation of the CYP7A1 and CYP8B1 genes by
-fetoprotein transcription factor (FTF), or mouse liver-related homology (LRH), NR5A2; see Refs. 15 and 30). However, FTF and hepatic nuclear factor-4
(HNF4
) differentially regulate rat and human CYP8B1 genes (44, 45). SHP interacts with FTF and inhibits rat CYP8B1 gene transcription (9) but interacts with HNF4
and inhibits the human CYP8B1 gene (45). In reporter assays, cotransfection with FXR enhances bile acid inhibition of the rat and human CYP7A1 gene (7) but has no effect on human CYP8B1 gene transcription (45). Also, bile acid feeding to Shp knockout mice reduces CYP7A1 and CYP8B1 mRNA levels as in wild-type mice (43). These results suggest that mechanisms alternative to the FXR/SHP pathway are involved in bile acid inhibition of the CYP7A1 and CYP8B1 genes and that the FXR/SHP pathway may not be the major mechanism for bile acid inhibition of CYP8B1 gene transcription. SHP-independent mechanisms, such as cytokine signaling pathways, may play a critical role in bile acid feedback inhibition of the CYP8B1 gene (5).
Bile acids have been shown to induce the release of inflammatory cytokines from the hepatic macrophages (Kupffer cells) present in the liver sinusoids. It has been suggested that cytokines may cross the sinusoidal membranes and act on the surrounding parenchymal cells to inhibit CYP7A1 activity and bile acid synthesis (33). Cytokine levels increase during hepatic inflammatory conditions such as sepsis and alcoholic, viral and autoimmune hepatitis, leading to intrahepatic cholestasis (10). Cytokines inhibit the genes encoding hepatobiliary transporters such as sodium taurocholate cotransport peptide (Ntcp, or Slc10a1; see Ref. 10), multidrug resistant protein 2 (mrp2 or Abcc2; see Ref. 11), mrp3 (Abcc3; see Ref. 2), and apical sodium-dependent bile acid transporter (Asbt or Slc10a2; see Ref. 4). Inhibition of bile acid synthesis and bile acid transport in the enterohepatic system may be an adaptive response to toxins and cholestatic liver injury. Bile acids are known to activate the mitogen-activated protein kinase (MAPK) signaling pathway that consists of a cascade of three protein kinases, leading to phosphorylation and activation of either c-Jun NH2-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), or p38 MAPK (3). Several lines of evidence show that bile acid inhibition of CYP7A1 gene transcription is mediated through the JNK pathway (17), which may inhibit the trans-activation potential of HNF4
, and result in inhibiting CYP7A1 gene transcription (8). Bile acids mimic phorbol esters that activate protein kinase C, leading to activation of c-Jun. It has been proposed that c-Jun either forms a repressive complex with an unknown factor, or activates SHP, to inhibit the CYP7A1 gene (17, 37). Another signaling cascade involving fibroblast growth factor (FGF) has been identified recently (19). In this pathway, FXR induces FGF-19, which activates FGF receptor 4 and the JNK pathway to inhibit bile acid synthesis. It is intriguing that this pathway is SHP independent and does not involve HNF4
or FTF.
We hypothesize that bile acids may inhibit the CYP8B1 gene via the MAPK signaling pathways. Also, because HNF4
is an important trans-activator of the human CYP8B1 gene, interleukin (IL-1
) may inhibit the HNF4
trans-activation of the CYP8B1 gene. In the current study, we demonstrated that IL-1
inhibited human CYP8B1 gene transcription and mRNA levels in human primary hepatocytes. We determined that the JNK pathway mediated this effect by inhibiting HNF4
gene expression and binding to the CYP8B1 gene. Our studies reveal a novel mechanism of cytokine inhibition of the human CYP8B1 gene transcription as a protection of hepatocytes from accumulating high levels of cholestatic bile acids, and an adaptive response to inflammatory agents and cholestasis.
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EXPERIMENTAL PROCEDURES
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Cell culture.
The human hepatoma cell line (HepG2) and the embryonic kidney cell line (HEK-293) were obtained from American Type Culture Collection (ATCC, Manassas, MA). The cells were grown in a 1:1 mixture of DMEM and F-12 (Life Technologies, Rockville, MD) supplemented with 100 U/ml penicillin G/streptomycin sulfate (Celox, Hopkins, MN) and 10% (vol/vol) heat-inactivated FCS (Irvine Scientific, Santa Ana, CA).
MTT assay.
Cytotoxicity of the human recombinant IL-1
(Preprotech) was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma Aldrich) calorimetric assay (14). HepG2 cells were seeded in 96-well plates and allowed to grow to confluence. Cells were then treated with increasing concentrations of IL-1
(0, 2, 5, and 10 ng/ml) in serum-containing media for 16 h. The medium was removed, and the cells were incubated with a solution containing 0.5 mg MTT/ml PBS (100 µl/well) at 37°C for 3 h. The MTT solution was removed, and the cells were overlaid with 50 µl/well of DMSO. The 96-well plate was then read on the plate reader (SpectraMax 250; Molecular Devices) at 570 nm using a reference wavelength of 690 nm.
Transient transfection assays.
Confluent cultures of HepG2 and HEK-293 cells grown in 24-well tissue culture plates were transiently transfected with plasmids by the lipofectamine method (Tfx20; Promega, Madison, WI). The luciferase (Luc) reporter constructs, expression plasmids, and pCMV
-galactosidase plasmid (one 1/10 of reporter plasmid, as internal control; Clontech) were transfected in each well. The pcDNA3 empty vector was added to normalize the amounts of DNA transfected in each assay. Cells were overlaid with media and were lysed 40 h after transfection. Cells were treated with 2 ng/ml IL-1
in fresh media 16 h before cell lysis. In the transfection assays using the inhibitors of the MAPK pathways, the cells were treated for 30 min with the specific inhibitors (Calbiochem, San Diego, CA) of the MAPK pathways, i.e., SP-600125 (JNK specific inhibitor, 25 µM), PD-98059 (ERK specific inhibitor, 20 µM), or SB-203580 (p38MAPK specific inhibitor, 25 µM) before treatment with IL-1
. Luciferase activity was assayed after cell lysis using the Luciferase Assay System (Promega), and luminescence was determined using a Lumat LB 9501 luminometer (Berthold Systems, Pittsburgh, PA). Luciferase activities were normalized for transfection efficiencies by dividing the relative light units (RLU) by
-galactosidase activity expressed from the cotransfected pCMV plasmid. The wild-type CYP8B1/Luc reporter p8B1-514/+300/Luc, 3'-deletion mutants of the CYP8B1/Luc reporter (p-514/+248, p-514/+220, p-514/+200, p-514/+76), and the p8B1mutant/Luc reporter that has the HNF4
binding site mutated were previously constructed (45). Mouse HNF4
/Luc reporter (pDGT43mHNF4pro744) containing 744 bp 5'-upstream sequence was provided by Dr. Todd Leff (Wayne State University, Detroit, MI). The expression plasmids were provided as follows: JNK1 (pcDNA3.1neo-HA-46
, Dr. James Woodgett, Ontario Cancer Institute, Toronto, Canada), dominant negative JNK1 (dnJNK, pcDNA3 FlagJNKapf, Dr. Roger Davis, Univ. of Massachusetts Medical School, Worcester, MA), c-Jun (Dr. Ronald Evans, Salk Institute, La Jolla, CA), dominant negative c-Jun (dnc-Jun, pcDNA3.1HisTAM67, Dr. Nancy Colburn, NCI, Frederick, MD), dominant negative HNF4
(pDGT23.1dnHNF4
-pcDNA3, Dr. Todd Leff, Wayne State University), HNF4
(originally from Dr. William Chin, Lilly Research Laboratories, Indianapolis, IN, subcloned to pcDNA3), and FTF (pCI FTF, Dr. L. Belanger, Le Centre de Recherche en Cancerologie de l'Universite Laval, Quebec, Canada). All assays were repeated at least three times. Reporter activities were expressed as RLU per
-gal activity. Statistical analyses were performed using the Student's t-test. A P value of <0.05 was considered as a statistically significant difference.
Quantitative real-time PCR.
Total RNA was isolated from primary human hepatocyte cells (case no. HH1088, pediatric female and case no. HH1165, pediatric female) provided by Dr. Steven Strom (University of Pittsburgh, Pittsburgh, PA) of the Liver Tissue Procurement and Distribution System and treated with increasing concentrations of IL-1
(0, 2, 5 and 10 ng/ml) or CDCA (0, 5, 10, 25, and 50 µM; Sigma Aldrich) for 16 h, using TRI-reagent (Sigma) according to the instruction manual. The RNA was then treated with RNase-free DNase I (DNA-free, DNase treatment kit; Ambion, Austin, TX) in 1x DNase reaction buffer for 1 h at 37°, after which the DNase was inactivated by incubation in the DNase inactivation solution for 2 min at room temperature. The concentration of the DNase I-treated total RNA was determined by ultraviolet spectrophotometry. Reverse transcription of DNase I-treated total RNA (2 µg) was performed using oligo(dT) primers (RETROscript first-strand synthesis kit for RT-PCR) to synthesize first-strand cDNA in a reaction volume of 20 µl. Primers for quantitative PCR of human CYP8B1 and human HNF4
were designed using the Primer Express software (Applied Biosystems, Foster City, CA) following the manufacturer's recommended parameters. The primer sequences are as follows: hCYP8B1F: 5'-GCCGACTCCAGCGTCTCTC-3'; hCYP8B1R: 5'-GCCCgccGTTGCTGAGCT-3'; hHNF4
: forward, 5'-GGGTGTCCATACGCATCCTT-3' and reverse, 5'-CATTGTCATCGATCTGCAGCT-3'. Primer sequences for human SHP (16), human CYP7A1 (32), and the human 23-kDa highly basic protein (used as the internal standard for normalizing RNA) were published previously (32). All primers were tested for specificity using a BLAST search. A 1x SYBR Green PCR master mix (Applied Biosystems) was used with 0.3 µM of forward and reverse primers in a total volume of 25 µl. PCR reactions contained 1 µl (100 ng) cDNA from the 20-µl RT reaction. To confirm that genomic DNA was absent, an aliquot of each RNA sample that had not been reverse transcribed was amplified using each primer pair. Also, to confirm the absence of any contamination in the reaction, controls were run in which H2O was substituted for a template for each primer set. All PCR reactions were done in triplicate. PCR amplification was performed as follows: 50°C for 2 min, 45 cycles (15 s each) at 95°C and 55°C for 1 min using a ABI PRISM 7700 sequence detector (Applied Biosystems). After PCR, the melting curve analysis was done using Dissociation Curves version 1 Ob1 Software (Applied Biosystems) to confirm the absence of primer dimers and to see if the correct product was amplified. Amplification data were analyzed using the Sequence Detector version 1.7 software (Applied Biosystems). Relative mRNA expression levels were calculated using the mathematical formulas (2
) recommended by Applied Biosystems (Applied Biosystems, User Bulletin no. 2, 1997). Statistical analysis of real-time PCR results were done using mean normalized cycle threshold (
Ct) values and the pooled standard deviation of the mean
Ct, which were analyzed by one-way ANOVA followed by Tukey's Honestly Significant post hoc test. A P value of <0.05 was considered as a statistically significant difference.
Immunoblot analysis.
Total cellular protein was extracted from confluent cultures of HepG2 cells treated with increasing concentrations of IL-1
(0, 2, 5 and 10 ng/ml) for 16 h. In the immunoblot assay using the inhibitors, the cells were treated for 30 min with the specific MAPK inhibitors SP-600125 (25 µM), PD-98059 (20 µM), or SB-203580 (25 µM) before treatment with IL-1
(5 ng/ml) for 16 h. The bottoms of the flasks were scraped with a cell scraper, and the cells were collected by centrifugation at 5,000 g for 5 min at 4°C. The pellet was then resuspended with 1x PBS and centrifuged again at 5,000 g for 5 min at 4°C. The resulting pellet was then suspended in 500 µl lysis buffer (62.5 mM Tris, 2 mM EDTA, 2.3% SDS, and 10% glycerol) containing the protease inhibitors, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 2 µg/ml each of leupeptin, pepstatin, and aprotinin. Protein concentration was determined by the bicinchoninic acid (BCA) protein assay kit (Pierce). The cellular extracts were stored as aliquots in 80°C. Total cellular protein (10 µg) was subjected to electrophoresis on a 10% SDS-polyacrylamide gel and then transferred to a nitrocellulose membrane [Hybond enhanced chemiluminescence (ECL); Amersham Pharmacia Biotech]. The membrane was blocked with 5% (wt/vol) nonfat dry milk in water overnight at 4°C and incubated with the HNF4
or actin antibody (Santa Cruz Biotechnology) at a dilution of 1:5,000 in TTBS (0.1% Tween 20 in Tris-buffered saline: 100 mM Tris·Cl, pH 7.5, and 0.9% NaCl) for 2 h at room temperature. Membranes were washed five times in TTBS and incubated with secondary antibody (horseradish peroxidase-conjugated anti-goat IgG) at a dilution of 1:3,000 in TTBS for 1 h at room temperature. Immunodetection was done with an ECL kit (Amersham Pharmacia Biotechnology). Membranes were imaged using Kodak Imaging Station 440.
Nuclear extracts.
Confluent cultures of HepG2 cells treated with IL-1
(0, 2, 5, and 10 ng/ml) for 16 h were lysed with trypsin and washed two times with cold PBS. The cells were then suspended in hypotonic buffer, swelled on ice for 10 min, and lysed using a Dounce homogenizer with a tight-fitting pestle. The homogenate was centrifuged for 30 s at 16,0000 g at 4°C after which the viscous nuclear pellet was lysed in buffer containing 0.4 M ammonium sulfate and centrifuged at 2°C for 90 min at 150,000 g to pellet nuclear debris and chromatin. Solid ammonium sulfate was added to precipitate the nuclear protein from the supernatant. The pellet was dissolved in nuclear dialysis buffer and dialyzed overnight at 4°C. Protein concentration was determined by the BCA protein assay kit (Pierce). The nuclear extracts were stored as aliquots in 80°C.
Electrophoretic mobility shift assay.
Synthetic oligonuleotide probe designed according to the sequences from +198 to +227 of the human CYP8B1 promoter was used in the electrophoretic mobility shift assay (EMSA; see Ref. 45). Double-stranded probes were prepared by heating equal molar amounts of complementary oligonucleotides to 95°C in 2 x 0.5 M NaCl and 15 mM sodium citrate, pH 7.0, and cooled to room temperature. The resulting double-stranded fragments were labeled by filling in the overhang with [
-32P]dCTP (3,000 Ci/mol) with the Klenow fragment of DNA polymerase I. The same oligonucleotide filled in with nonlabeled dNTPs was used as a cold competitor. Labeled fragments were purified through two G-50 spin columns. The binding reaction consisted of the addition of 1 µg nuclear extracts to 50,000 cpm of labeled oligonucleotide probe dissolved in 25 µl binding buffer [12 mM HEPES, pH 7.9, 50 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, 15% glycerol, and 1 µg poly(dI-dC)]. Samples were incubated for 20 min at room temperature. Polyacrylamide gel (40%) was prepared and prerun for 1 h at 100 volts. Electrophoresis was performed at room temperature at a constant 200 volts for 1.5 h. The gel was dried and autoradiographed using Phosphoimager 445Si (Molecular Dynamics, Sunnyvale, CA). The images were analyzed using IP Lab Gel software (Signal Analytics, Vienna, VA). Antibody supershift assay was carried out by adding 2 µl of the antibody against HNF4
(Santa Cruz Biotechnology, Santa Cruz, CA) to the nuclear extract and incubated for 15 min before adding the labeled probe.
Chromatin immunoprecipitation assay.
HepG2 cells were grown in 100-mm culture dishes to 80% confluence. The cells were then treated with 10 ng/ml IL-1
or 25 µM CDCA for 16 h. Chromatin immunoprecipitation (ChIP) assays were performed using a ChIP Assay kit (Upstate Cell Signaling Solutions, Lake Placid, NY) according to the manufacturer's protocol. Briefly, HepG2 cells were cross-linked in 1% formaldehyde for 10 min and washed two times with ice-cold PBS containing protease inhibitors (1 mM PMSF, 1 µg/ml aprotinin, and 1 µg/ml pepstanin A; Sigma Aldrich) two times. Cells were scraped and incubated in 1% SDS lysis buffer (1% SDS, 10 mM EDTA, and 50 mM Tris·HCl, pH 8.1) for 10 min on ice and sonicated using a Branson sonifier 250 with a micro tip (setting 6 for 15 s at 40% output) to break the DNA into 0.2- to 2-kb fragments. Cell lysates were collected by centrifugation and diluted 10-fold in ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris·HCl, pH 8.1, and 167 mM NaCl). Cell lysate solution (5%) in ChIP dilution buffer was kept aside as input. After preclearing the remaining diluted cell lysate with protein A-agarose, DNA-protein complexes were precipitated by incubating the cell lysates with 10 µg HNF4
antibody or nonimmune antibody (Santa Cruz Biotechnology) overnight followed by incubation with protein A-agarose beads for 3 h. The beads were washed with one time each with low-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris·HCl, pH 8.1, and 150 mM NaCl), high-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris·HCl, pH 8.1, and 500 mM NaCl), and LiCl wash buffer (0. 25 M LiCl, 1% Nonidet P-40, 1% deoxycholate, 1 mM EDTA, and 10 mM Tris·HCl, pH 8.1) and two times with 10 mM Tris·HCl and 1 mM EDTA, pH 8.0. Samples were eluted two times with freshly prepared ChIP elution buffer (1% SDS and 0.1 M NaHCO3), and the eluants were combined. Reverse cross-linking of both the eluant and the input fractions was achieved by adjusting the NaCl concentration to 200 mM and incubating at 65°C for 4 h. The eluants were then incubated at 45°C for 1 h in 0.04 µg/µl proteinase K. DNA was extracted using phenol/chloroform and precipitated using isopropanol. A 251-bp DNA fragment containing the bile acid response element of the CYP8B1 promoter was PCR amplified for 45 cycles (primer sequence given below) using 5 µl of the DNA as template and analyzed on a 1.5% agarose gel. PCR primers for amplifying a 251-bp DNA fragment containing the bile acid response element of the CYP8B1 promoter were as follows: L8B1ChIPHNF4F: 5'-AAGCTGGTGAGCAGCTGTGA-3' and L8B1ChIPHNF4R: 5'-CACACTGTTCCCTGGGTGC-3'.
Glutathione S-transferase fusion protein expression.
The pGEX-4T-1 plasmid encoding the glutathione S-transferase (GST) protein alone was obtained from Amersham Pharmacia Biotech, and the pET23a-GST-HNF4
plasmid encoding a fusion protein of GST with full-length human HNF4
was a kind gift from Todd Leff (Wayne State University). Both plasmids were transformed into Escherichia coli BL21 cells (Amersham Pharmacia Biotech). Cells were grown to an OD of
0.6 and induced with 1 mM isopropyl thio-
-galactoside (IPTG Amersham Pharmacia Biotechnologies) for an additional 2 h. Cells were centrifuged, and the bacterial pellets were suspended in Triton X-100 lysis buffer (0.05 M Tris·HCl, 5 mM EDTA, 0.05 M NaCl, 0.1% Triton X-100, and 10% glycerol) containing the protease inhibitors (1 mM PMSF, 1 µg/ml aprotinin, and 1 µg/ml pepstanin A; Sigma) and sonicated 10 times in pulses of 30 s each. Cell lysates were clarified by centrifugation at 10,000 g for 10 min at 4°C. Cell lysates (1 ml each) were incubated with immobilized glutathione beads (Amersham Pharmacia Biotechnologies) at 4°C for 2 h. The beads were washed four times in the same lysis buffer, and the proteins bound to the glutathione beads were eluted with an elution buffer (50 mM Tris·HCl, pH 8.0) containing 10 mM reduced glutathione. The protein samples, mixed with 2x protein loading buffer, were loaded on an SDS-PAGE. The gel was then stained with Coomassie Blue stain (Bio-Rad) to confirm the correct protein expressions. Protein concentration was determined by the Coomassie Plus-Better Bradford Assay Reagent (Pierce). The proteins were stored as aliquots in 80°C for future use in the kinase assays.
In vitro kinase assay.
In the JNK assays, 4 µg GST, 4 µg GST-HNF4
, or 2.5 µg activation transcription factor 2 (ATF2) protein (Upstate Cell Signaling Solutions) were incubated at 37°C for 30 min with 20 ng active JNK1 (Upstate Cell Signaling Solutions) along with 10 µCi [
-32P]ATP in the JNK kinase reaction buffer [500 mM Tris·HCl pH 7.5, 1% 2-mercaptoethanol, 20 mM MOPS, pH 7.2, 25 mM
-glycerol phosphate, 5 mM EGTA, 1 mM sodium orthovandate, and 1 mM dithiothreitol (DTT)] to which 75 mM MgCl2 and 500 µM ATP were added. An enzyme-negative reaction was performed with each protein without adding active JNK1. In the AMP kinase (AMPK) assays, 4 µg GST, 4 µg GST-HNF4
, or 100 mM serine alanine methionine serine (SAMS) peptide (Upstate Cell Signaling Solutions) was incubated at 37°C for 30 min with 1,000 mU of AMPK (Upstate Cell Signaling Solutions) along with 10 µCi [
-32P]ATP in the AMPK kinase reaction buffer (20 mM HEPES-NaOH, pH 7.0, 0.01% Brij-35, 20 mM MOPS, pH 7.2, 25 mM
-glycerol phosphate, 5 mM EGTA, 1 mM sodium orthovandate, and 1 mM DTT either with or without 300 µM AMP) to which 75 mM MgCl2 and 500 µM cold ATP were added. An enzyme-negative reaction was performed with each protein without adding AMPK. After incubation, all samples were mixed with Laemmli buffer, boiled for 5 min, and analyzed by SDS-PAGE. The gel was dried and autoradiographed.
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RESULTS
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Effect of IL-1
on CYP8B1 mRNA expression in human primary hepatocytes.
We performed quantitative real-time PCR to determine the effect of IL-1
on CYP8B1 mRNA expression in human primary hepatocytes. Treatment of increasing amounts of IL-1
for 16 h markedly reduced CYP8B1 mRNA levels in human primary hepatocytes by
7090% (Table 1). Similarly, IL-1
reduced CYP7A1 mRNA expression in a dose-dependent manner. Interestingly, IL-1
also reduced HNF4
and SHP mRNA levels in human hepatocytes by >80% at 10 ng/ml. To compare the effect of IL-1
with bile acids on mRNA expression, we treated human primary hepatocytes with CDCA, a FXR ligand. As shown in Table 2, CDCA significantly inhibited CYP8B1, CYP7A1, and HNF4
mRNA levels in a dose-dependent manner, at the physiological concentrations (up to 25 µM) by
4070%. In contrast, CDCA dose-dependently induced SHP mRNA levels by up to fourfold at 50 µM. These data revealed that both IL-1
and CDCA reduced CYP8B1, CYP7A1, and HNF4
mRNA expression levels but had different effects on SHP mRNA expression in human primary hepatocytes. Previously, we reported that CDCA feeding (1%) reduced HNF4
mRNA expression in rat livers (44), and CDCA reduced HNF4
protein expression and gene transcription (45).
Effect of IL-1
on human CYP8B1 gene transcription.
To determine the effect of IL-1
on CYP8B1 gene transcription, reporter assays were performed in HepG2 cells transiently transfected with the human CYP8B1/Luc reporter plasmid, p8B1514/+300/Luc, and treated with IL-1
(2 ng/ml) for 16 h. The cells were tested for viability and cell functionality on exposure to IL-1
by MTT assay. IL-1
did not show any signs of toxicity up to the highest concentration tested (i.e., 10 ng/ml; data not shown). IL-1
inhibited the CYP8B1 reporter activity by
80% (Fig. 1A). The same experiment was repeated in the HEK-293 cells. Figure 1B shows that IL-1
did not alter the CYP8B1 reporter activity in HEK-293 cells. These results suggest that liver-specific factors may be required for the inhibitory effect of IL-1
on CYP8B1 gene transcription.
Identification of the IL-1
-activated MAPK signaling pathway involved in inhibition of CYP8B1 gene transcription.
Cytokines are known to activate MAPK signaling pathways. To determine which of the three MAPK pathways are involved in IL-1
inhibition of CYP8B1 gene transcription, the specific inhibitor of JNK, ERK, or p38MAPK was added in HepG2 cells transfected with p8B1-514/+300/Luc reporter, with or without prior treatment with IL-1
. Preincubation with the JNK inhibitor SP-600125 (25 µM) completely blocked the inhibitory effect of IL-1
on the CYP8B1 reporter activity (Fig. 2A). However, ERK inhibitor PD-98059 (20 µM) and p38MAPK inhibitor SB-203580 (25 µM) had no such effect on CYP8B1 reporter activity (Fig. 2A). These results suggest that only the JNK pathway is involved in mediating the inhibitory effect of IL-1
on CYP8B1 gene transcription. To further study the role of the JNK pathway in the suppression of CYP8B1 gene transcription by IL-1
, expression plasmids for JNK1 were cotransfected with CYP8B1 reporter in HepG2 cells. As shown in Fig. 2B, ectopically expressed JNK1 inhibited the CYP8B1 basal promoter activity, whereas dnJNK1 did not. When c-Jun, a target of JNK1, was cotransfected, the reporter activity was markedly inhibited, whereas dnc-Jun markedly stimulated CYP8B1 reporter activity. These results suggest that endogenous c-Jun in HepG2 cells might inhibit CYP8B1 gene transcription. Thus any upregulation of JNK1 or c-Jun will inhibit CYP8B1 gene transcription. These results further support the finding that the JNK pathway of the MAPK signaling cascade is responsible for mediating the IL-1
inhibition of CYP8B1 gene transcription.
Mapping the IL-1
-responsive sequence on the CYP8B1 gene.
To map the DNA sequence on the CYP8B1 gene that is responsive to IL-1
, transient transfection assay was performed using 3'-deletion mutants of the CYP8B1/Luc reporter. The inhibitory effect of IL-1
on CYP8B1/Luc reporter activity was diminished when the region from +220 to +200 was deleted (Fig. 3A). This IL-1
-responsive region contains an overlapping direct repeat with 1 base spacing (DR-1) and FTF binding site that was previously shown to bind HNF4
and FTF, respectively (45). To investigate the possible roles of HNF4
and/or FTF on mediating the inhibitory effect of IL-1
on the CYP8B1 gene, we mutated the HNF4
binding site but created an FTF binding site on the reverse strand of the CYP8B1 gene (p8B1mutant/Luc) and assayed the reporter activity in HepG2 cells. As shown in Fig. 3B, mutation of the HNF4
binding site markedly reduced the basal reporter activity, and IL-1
did not inhibit the mutant reporter activity. This result suggests that the HNF4
binding site is required for mediating IL-1
inhibition of CYP8B1 gene transcription. On the other hand, FTF does not play a role in mediating the IL-1
effect because this mutant reporter has a FTF binding site and does bind FTF (data not shown). To further corroborate the role of HNF4
in mediating the IL-1
effect, dnHNF4
was cotransfected in HepG2 cells to test its effect on reporter activity. Figure 3C shows that dnHNF4
strongly inhibited basal reporter activity. This dnHNF4
has a defective DNA binding domain but is able to dimerize with wild-type HNF4
(38), thus markedly reducing CYP8B1 gene expression. Addition of IL-1
did not further reduce the reporter activity. These results support our finding that HNF4
is critical for trans-activating the CYP8B1 gene, and IL-1
inhibits HNF4
induction of human CYP8B1 gene transcription. Furthermore, overexpression of HNF4
stimulated the human CYP8B1 promoter activity as expected; however, IL-1
was not able to suppress the CYP8B1 promoter activity stimulated by HNF4
(Fig. 3D). As a negative control, IL-1
was able to suppress CYP8B1 reporter activity in cells overexpressing FTF. These data further support that HNF4
is involved in IL-1
inhibition of the CYP8B1 gene.
Effects of IL-1
on HNF4
gene expression.
To determine if IL-1
had any direct effect on HNF4
gene transcription, the HNF4
/Luc reporter was transfected in HepG2 cells, which were subsequently treated with IL-1
. Figure 4A shows that IL-1
decreases the HNF4
reporter activity. These results are consistent with IL-1
inhibition of HNF4
mRNA expression in primary hepatocytes and suggested that inhibition of HNF4
gene transcription by IL-1
may be responsible for the suppression of CYP8B1 by IL-1
. Because IL-1
treatment decreased HNF4
reporter activity and mRNA levels, it was interesting to see if it also alters the HNF4
protein levels. HepG2 cells were treated with increasing concentrations of IL-1
, and immunoblot analysis with HNF4
antibody was performed to estimate the amount of HNF4
protein in the control and treated cells. The amount of HNF4
protein in HepG2 cells decreased in a dose-dependent manner by IL-1
(Fig. 4B). Intracellular actin levels were determined as an internal control. Thus it can be concluded that IL-1
inhibition of HNF4
transcriptional activity leads to a decrease in HNF4
mRNA and protein levels and is responsible for the suppression of CYP8B1 gene transcription.
Effect of IL-1
on HNF4
binding to the CYP8B1 gene.
We reported previously that HNF4
bound to the DR-1 element on the human CYP8B1 promoter (45). To determine if IL-1
treatment alters the ability of HNF4
to bind to the DR-1 element, EMSA of an oligonucleotide probe based on the DR-1 element of the human CYP8B1 gene was performed using nuclear extracts prepared from HepG2 cells treated with increasing concentrations of IL-1
. As shown in Fig. 5A, increasing concentrations of IL-1
decreased the HNF4
binding to the probe. It is likely that IL-1
treatment reduced nuclear HNF4
protein or its DNA binding activity. This result is consistent with IL-1
inhibition of HNF4
gene transcription shown in Fig. 4A and supports the hypothesis that IL-1
reduces HNF4
trans-activation of the CYP8B1 gene. To confirm that IL-1
reduces HNF4
binding to the CYP8B1 promoter in cells, ChIP assay was performed. HepG2 cell extracts (control and IL-1
treated) were immunoprecipitated with HNF4
antibody, and the amount of the immunoprecipitated DNA fragments containing the HNF4
binding site on the CYP8B1 promoter was quantified by PCR. As shown in Fig. 5B, IL-1
treatment reduces the amount of HNF4
bound to the CYP8B1 chromatin, as indicated by reduced levels of anti-HNF4
antibody-precipitated chromatins. Also, 25 µM CDCA reduced the HNF4
bound to the CYP8B1 chromatin. Nonimmune IgG was used as a negative control of ChIP assay. These data are consistent with the real-time RT-PCR results (Tables 1 and 2). Thus both the EMSA and ChIP assay results suggest that IL-1
reduces HNF4
binding to the CYP8B1 chromatin.
Effect of the MAPK inhibitors on IL-1
-mediated inhibition of HNF4
protein expression.
To determine if the JNK pathway had any effect on HNF4
protein expression levels, we performed an immunoblot assay. HepG2 cells were preincubated for 30 min with the specific inhibitors of JNK, ERK, or p38MAPK before treatment with IL-1
(5 ng/ml) for 16 h. Pretreatment with the JNK specific inhibitor SP-600125 (25 µM) was able to block the IL-1
-mediated suppression of HNF4
protein levels (Fig. 6). However, treatment with the ERK-specific inhibitor PD-98059 (20 µM) and the p38 MAPK-specific inhibitor SB-203580 (25 µM) did not block IL-1
suppression of HNF4
protein levels. The results suggest that the JNK pathway mediates the effect of IL-1
on the CYP8B1 promoter, possibly by regulation of HNF4
protein expression levels.
JNK1 phosphorylation of HNF4
.
It is known that HNF4
is phosphorylated by protein kinase A (39), ERK (34), and AMPK (20, 26) and that phosphorylated HNF4
loses its DNA-binding activity (25). Because the JNK was shown to be activated in HepG2 cells by IL-1
(28), we were interested to see if JNK1 could phosphorylate HNF4
. Purified GST-tagged HNF4
fusion protein was incubated with active JNK1 in the presence of [
-32P]ATP. JNK1 could phosphorylate the GST-HNF4
fusion protein but did not phosphorylate the GST protein (Fig. 7). Without JNK, HNF4
was not phosphorylated. As a positive control for JNK1 phosphorylation, JNK1 strongly phosphorylated ATF2. We also did AMPK phosphorylation of HNF4
. As shown in Fig. 7, bottom, AMP activated AMPK-phosphorylated HNF4
and the positive control, SAMS peptide. Without AMPK, HNF4
and SAMS were not phosphorylated. These results suggest that JNK1 was able to phosphorylate HNF4
and also contribute to reduced binding of HNF4
to DNA upon IL-1
treatment (Fig. 5A).
 |
DISCUSSION
|
---|
Using the specific inhibitors of the MAPK pathway, we were able to delineate the JNK pathway as the cytokine signaling pathway that inhibits CYP8B1 gene transcription. We also demonstrated that JNK1 and c-Jun markedly decreased CYP8B1 gene transcription and that dnJNK1 and c-Jun blocked the inhibition. This is similar to the previous finding that bile acids activate c-Jun, which inhibits CYP7A1 mRNA expression in rat primary hepatocytes (37). Our previous studies show that HNF4
is a strong activator of the human CYP8B1 gene and that the HNF4
binding site mediates bile acid inhibition (45). In this study, we found that IL-1
markedly reduced CYP8B1 and HNF4
mRNA levels in human primary hepatocytes. These results led us to hypothesize that IL-1
might alter the levels of HNF4
gene expression and result in inhibition of CYP8B1 gene transcription. Our mutagenesis analyses provide strong evidence that the bile acid response element is a cytokine response element on the CYP8B1 gene. The finding that a dnHNF4
inhibits the CYP8B1 gene and abrogates the inhibitory effect of IL-1
on CYP8B1 gene transcription supports that HNF4
is involved in IL-1
inhibition of the human CYP8B1 gene. Our results that IL-1
inhibits the HNF4
reporter activity, protein levels, and HNF4
binding to the CYP8B1 chromatin further support our model that HNF4
is a downstream target of the cytokine-JNK pathway and that the cytokine inhibits HNF4
trans-activation of the CYP8B1 gene. The results that JNK-specific inhibitor blocked IL-1
inhibition of HNF4
expression and JNK1 was able to phosphorylate HNF4
suggest that the JNK pathway inhibits HNF4
expression and reduces HNF4
binding to DNA and results in downregulation of the human CYP8B1 gene. Phosphorylation of nuclear receptors by the MAPK pathway has been reported (27, 28, 35).
In contrast to the strong induction of SHP mRNA expression by CDCA, IL-1
markedly reduced SHP mRNA expression in human primary hepatocytes. This result suggests that the inhibitory effect of IL-1
on the CYP8B1 gene does not involve SHP because inhibition of a negative factor would induce CYP8B1 gene transcription. We reported previously that SHP could interact with HNF4
and inhibit human CYP8B1 gene transcription (45). However, the SHP interaction with HNF4
is much weaker than with FTF. Furthermore, bile duct ligation alone reduced CYP8B1, induced CYP7A1, but did not have any effect on Shp mRNA expression levels. In
-naphthylisothiocyanate-induced intrahepatic cholestatic rats, CYP8B1 mRNA expression is reduced by 80%, but CYP7A1 and Shp mRNA expression is not altered (29). Also, CYP8B1 mRNA expression is not altered in a recent study of bile acid metabolism in Fxr null mice (24). These results support our conclusion that the FXR-SHP pathway may not play a major role in bile acid inhibition of CYP8B1 gene transcription. It is clear that redundant mechanisms are involved in bile acid inhibition of CYP7A1 and CYP8B1 and explain the finding that CYP7A1 and CYP8B1 mRNA expression remain inhibited by bile acid feeding in Shp null mice (43). It is intriguing that CYP8B1, but not CYP7A1, is reexpressed in the Shp null mice fed a diet containing CA or CA plus cholesterol for 12 wk (42). All these results suggest that CYP7A1 and CYP8B1 genes are regulated by somewhat different mechanisms by bile acids.
Shp gene transcription is under a complex regulation by many transcription factors, including FXR, activation protein 1, liver orphan receptor
(LXR
), estrogen receptor-
, sterol response element binding protein-1c (SREBP-1c), and FTF (5, 16, 22). In this study, we found that IL-1
markedly reduced SHP mRNA levels in primary human hepatocytes. Interestingly, IL-1
inhibited Shp gene transcription, and a putative HNF4
binding site (563-TGGACAGTGGGCA-551) is located in the human but not mouse Shp promoter (data not shown). It is likely that IL-1
inhibition of the HNF4
gene also results in inhibition of Shp gene transcription in human liver. Species differences in LXR
and SREBP-1c regulation of CYP7A1, CYP8B1, and Shp genes is well documented (5, 16, 22). It is possible that IL-1
inhibition of the Shp gene may be also an adaptive response to inflammation and cholestasis. Inhibition of this negative nuclear receptor may promote nuclear receptor activation of gene transcription during hepatocyte regeneration and differentiation.
Previous studies have reported that lipopolysaccharide (LPS) inhibits CYP7A1 and sterol 27-hydroxylase gene transcription and also retinoid X receptor (R x R), FTF, and FXR expression in hamster and mouse livers (1, 13, 23, 31). Unpublished results show that LPS also reduces CYP8B1 mRNA levels in mice (K. R. Feingold and J. Y. L. Chiang). Endotoxin and cytokine inhibition of gene transcription is an acute-phase response that decreases bile acid synthesis, secretion, and transport in the endohepatic system to protect the liver from accumulating inflammatory and toxic agents. TNF-
and IL-1
levels are increased after bile duct ligation and during cholestasis and liver injury. Inhibition of bile acid synthesis by cytokines may also contribute to hypercholesterolemia observed in Syrian hamsters treated with LPS, TNF-
, or IL-1
(12, 18). An increase in cholesterol synthesis and reduction of bile acid synthesis may contribute to hypercholesterolemia and hypertriglyceridemia observed in the acute-phase response and inflammatory conditions.
In summary, our study suggests that cytokines inhibit CYP8B1 gene transcription via activation of the JNK pathway, which reduces HNF4
gene transcription and its DNA-binding activity and results in inhibition of human CYP8B1 gene transcription. Inhibition of bile acid synthesis and sinusoid bile acid uptake is an adaptive response to inflammation and a protection of the liver from accumulating toxic bile acids and developing intrahepatic cholestasis.
 |
GRANTS
|
---|
This study is supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-44442 and DK-58379. A. Jahan is a recipient of an American Heart Association Ohio Valley affiliates Predoctoral Fellowship.
 |
FOOTNOTES
|
---|
Address for reprint requests and other correspondence: J. Y. L. Chiang, Dept. of Biochemistry and Molecular Pathology, Northeastern Ohio Universities College of Medicine, 4209 State Route 44, P. O Box 95, Rootstown, OH 44272 (E-mail: jchiang{at}neoucom.edu)
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.
 |
REFERENCES
|
---|
- Beigneux AP, Moser AH, Shigenaga JK, Grunfeld C, and Feingold KR. The acute phase response is associated with retinoid X receptor repression in rodent liver. J Biol Chem 275: 1639016399, 2000.[Abstract/Free Full Text]
- Bohan A, Chen WS, Denson LA, Held MA, and Boyer JL. Tumor necrosis factor alpha-dependent up-regulation of Lrh-1 and Mrp3(Abcc3) reduces liver injury in obstructive cholestasis. J Biol Chem 278: 3668836698, 2003.[Abstract/Free Full Text]
- Chang L and Karin M. Mammalian MAP kinase signalling cascades. Nature 410: 3740, 2001.[CrossRef][ISI][Medline]
- Chen F, Ma L, Sartor RB, Li F, Xiong H, Sun AQ, and Shneider B. Inflammatory-mediated repression of the rat ileal sodium-dependent bile acid transporter by c-fos nuclear translocation. Gastroenterology 123: 20052016, 2002.[CrossRef][ISI][Medline]
- Chiang JY. Bile acid regulation of hepatic physiology. III. Bile acids and nuclear receptors. Am J Physiol Gastrointest Liver Physiol 284: G349G356, 2003.[Abstract/Free Full Text]
- Chiang JYL. Regulation of bile acid synthesis. Front Biosci 3: D176D193, 1998.[Medline]
- Chiang JYL, Kimmel R, Weinberger C, and Stroup D. FXR responds to bile acids and represses cholesterol 7
-hydroxylase gene (CYP7A1) transcription. J Biol Chem 275: 1091810924, 2000.[Abstract/Free Full Text]
- De Fabiani E, Mitro N, Anzulovich AC, Pinelli A, Galli G, and Crestani M. The negative effects of bile acids and tumor necrosis factor
on the transcription of cholesterol 7
-hydroxylase gene (CYP7A1) converge to hepatic nuclear factor-4. A novel mechanism of feedback regulation of bile acid synthesis mediated by nuclear receptors. J Biol Chem 276: 3070830716, 2001.[Abstract/Free Full Text]
- Del Castillo-Olivares A and Gil G. Suppression of sterol 12
-hydroxylase transcription by the short heterodimer partner: insights into the repression mechanism. Nucleic Acids Res 29: 40354042, 2001.[Abstract/Free Full Text]
- Denson LA, Auld KL, Schiek DS, McClure MH, Mangelsdorf DJ, and Karpen SJ. Interleukin-1
suppresses retinoid transactivation of two hepatic transporter genes involved in bile formation. J Biol Chem 275: 88358843, 2000.[Abstract/Free Full Text]
- Denson LA, Bohan A, Held MA, and Boyer JL. Organ-specific alterations in RAR
: RXR
abundance regulate rat Mrp2 (Abcc2) expression in obstructive cholestasis. Gastroenterology 123: 599607, 2002.[CrossRef][ISI][Medline]
- Feingold KR, Pollock AS, Moser AH, Shigenaga JK, and Grunfeld C. Discordant regulation of proteins of cholesterol metabolism during the acute phase response. J Lipid Res 36: 14741482, 1995.[Abstract]
- Feingold KR, Spady DK, Pollock AS, Moser AH, and Grunfeld C. Endotoxin, TNF, and IL-1 decrease cholesterol 7
-hydroxylase mRNA levels and activity. J Lipid Res 37: 223228, 1996.[Abstract]
- Gerlier D and Thomasset N. Use of MTT colorimetric assay to measure cell activation. J Immunol Methods 94: 5763, 1986.[CrossRef][ISI][Medline]
- Goodwin B, Jones SA, Price RR, Watson MA, McKee DD, Moore LB, Galardi C, Wilson JG, Lewis MC, Roth ME, Maloney PR, Willson TM, and Kliewer SA. A regulatory cascade of the nuclear receptors FXR, SHP-1, and LRH-1 represses bile acid biosynthesis. Mol Cell 6: 517526, 2000.[CrossRef][ISI][Medline]
- Goodwin B, Watson MA, Kim H, Miao J, Kemper JK, and Kliewer SA. Differential regulation of rat and human CYP7A1 by the nuclear oxysterol receptor liver X receptor-
. Mol Endocrinol 17: 386394, 2003.[Abstract/Free Full Text]
- Gupta S, Stravitz RT, Dent P, and Hylemon PB. Down-regulation of cholesterol 7
-hydroxylase (CYP7A1) gene expression by bile acids in primary rat hepatocytes is mediated by the c-Jun N-terminal kinase pathway. J Biol Chem 276: 1581615822, 2001.[Abstract/Free Full Text]
- Hardardottir I, Moser AH, Memon R, Grunfeld C, and Feingold K. Effects of TNF, IL-1, and the combination of both cytokines on cholesterol metabolism in Syrian hamsters. Lymphokine Cytoline Res 13: 161166, 1994.
- Holt JA, Luo G, Billin AN, Bisi J, McNeill YY, Kozarsky KF, Donahee M, Wang Da Y, Mansfield TA, Kliewer SA, Goodwin B, and Jones SA. Definition of a novel growth factor-dependent signal cascade for the suppression of bile acid biosynthesis. Genes Dev 17: 15811591, 2003.[Abstract/Free Full Text]
- Hong YH, Varanasi US, Yang W, and Leff T. AMP-activated protein kinase regulates HNF4
transcriptional activity by inhibiting dimer formation and decreasing protein stability. J Biol Chem 278: 2749527501, 2003.[Abstract/Free Full Text]
- Ishida H, Yamashita C, Kuruta Y, Yoshida Y, and Noshiro M. Insulin is a dominant suppressor of sterol 12
-hydroxylase P450 (CYP8B) expression in rat liver: possible role of insulin in circadian rhythm of CYP8B. J Biochem (Tokyo) 127: 5764, 2000.[Abstract]
- Kim HJ, Kim JY, Park SK, Seo JH, Kim JB, Lee IK, Kim KS, and Choi HS. Differential regulation of human and mouse orphan nuclear receptor small heterodimer partner promoter by sterol regulatory element binding protein-1. J Biol Chem 279: 2812228131, 2004.[Abstract/Free Full Text]
- Kim MS, Shigenaga J, Moser A, Feingold K, and Grunfeld C. Repression of farnesoid X receptor during the acute phase response. J Biol Chem 278:89888995, 2003.[Abstract/Free Full Text]
- Kok T, Hulzebos CV, Wolters H, Havinga R, Agellon LB, Stellaard F, Shan B, Schwarz M, and Kuipers F. Enterohepatic circulation of bile salts in farnesoid X receptor-deficient mice: efficient intestinal bile salt absorption in the absence of ileal bile acid-binding protein. J Biol Chem 278: 4193041937, 2003.[Abstract/Free Full Text]
- Ktistaki E, Ktistaki T, Papadogeorgaki E, and Talianidis I. Recruitment of hepatocyte nuclear factor 4 into specific intranuclear compartiments depends on tyrosine phosphorylation that affects its DNA-binding and transactivation potential. Proc Natl Acad Sci USA 92: 98769880, 1995.[Abstract/Free Full Text]
- Leclerc I, Lenzner C, Gourdon L, Vaulont S, Kahn A, and Viollet B. Hepatocyte nuclear factor-4
involved in type 1 maturity-onset diabetes of the young is a novel target of AMP-activated protein kinase. Diabetes 50: 15151521, 2001.[Abstract/Free Full Text]
- Lee HY, Suh YA, Robinson MJ, Clifford JL, Hong WK, Woodgett JR, Cobb MH, Mangelsdorf DJ, and Kurie JM. Stress pathway activation induces phosphorylation of retinoid X receptor. J Biol Chem 275: 3219332199, 2000.[Abstract/Free Full Text]
- Li D, Zimmerman TL, Thevananther S, Lee HY, Kurie JM, and Karpen SJ. Interleukin-1
-mediated suppression of RXR: RAR transactivation of the Ntcp promoter is JNK-dependent. J Biol Chem 277: 3141631422, 2002.[Abstract/Free Full Text]
- Liu Y, Binz J, Numerick MJ, Dennis S, Luo G, Desai B, MacKenzie KI, Mansfield TA, Kliewer SA, Goodwin B, and Jones SA. Hepatoprotection by the farnesoid X receptor agonist GW4064 in rat models of intra- and extrahepatic cholestasis. J Clin Invest 112: 16781687, 2003.[Abstract/Free Full Text]
- Lu TT, Makishima M, Repa JJ, Schoonjans K, Kerr TA, Auwerx J, and Mangelsdorf DJ. Molecular basis for feedback regulation of bile acid synthesis by nuclear receptors. Mol Cell 6: 507515, 2000.[CrossRef][ISI][Medline]
- Memon RA, Moser AH, Shigenaga JK, Grunfeld C, and Feingold KR. In vivo and in vitro regulation of sterol 27-hydroxylase in the liver during the acute phase response. Potential role of hepatocyte nuclear factor-1. J Biol Chem 276: 3011830126, 2001.[Abstract/Free Full Text]
- Menke JG, Macnaul KL, Hayes NS, Baffic J, Chao YS, Elbrecht A, Kelly LJ, Lam MH, Schmidt A, Sahoo S, Wang J, Wright SD, Xin P, Zhou G, Moller DE, and Sparrow CP. A novel liver X receptor agonist establishes species differences in the regulation of cholesterol 7
-hydroxylase (CYP7a). Endocrinology 143: 25482558, 2002.[Abstract/Free Full Text]
- Miyake JH, Wang SL, and Davis RA. Bile acid induction of cytokine expression by macrophages correlates with repression of hepatic cholesterol 7
-hydroxylase. J Biol Chem 275: 2180521808, 2000.[Abstract/Free Full Text]
- Reddy S, Yang W, Taylor DG, Shen X, Oxender D, Kust G, and Leff T. Mitogen-activated protein kinase regulates transcription of the ApoCIII gene. Involvement of the orphan nuclear receptor HNF4. J Biol Chem 274: 3305033056, 1999.[Abstract/Free Full Text]
- Rochette-Egly C. Nuclear receptors: integration of multiple signalling pathways through phosphorylation. Cell Signal 15: 355366, 2003.[CrossRef][ISI][Medline]
- Sinal CJ, Tohkin M, Miyata M, Ward JM, Lambert G, and Gonzalez FJ. Targeted disruption of the nuclear receptor FXR/BAR impairs bile acid and lipid homeostasis. Cell 102: 731744, 2000.[CrossRef][ISI][Medline]
- Stravitz RT, Vlahcevic ZR, and Hylemon PB. Signal transduction pathways regulating cholesterol 7
-hydroxylase transcription by bile acids. In: Bile Acids and Cholestasis, edited by Paumgartner G, Stiehl A, Gerok W, Keppler D, and Leuschner U. Boston, MA: Kluwer, 1999, p. 3950.
- Taylor DG, Haubenwallner S, and Leff T. Characterization of a dominant negative mutant form of the HNF-4 orphan receptor. Nucleic Acids Res 24: 29302935, 1996.[Abstract/Free Full Text]
- Viollet B, Kahn A, and Raymondjean M. Protein kinase A-dependent phosphorylation modulates DNA-binding activity of hepatocyte nuclear factor 4. Mol Cell Biol 17: 42084219, 1997.[Abstract]
- Vlahcevic ZR, Eggertsen G, Bjorkhem I, Hylemon PB, Redford K, and Pandak WM. Regulation of sterol 12
-hydroxylase and cholic acid biosynthesis in the rat. Gastroenterology 118: 599607, 2000.[ISI][Medline]
- Wang DQ, Paigen B, and Carey MC. Phenotypic characterization of Lith genes that determine susceptibility to cholesterol cholelithiasis in inbred mice: physical-chemistry of gallbladder bile. J Lipid Res 38: 13951411, 1997.[Abstract]
- Wang L, Han Y, Kim CS, Lee YK, and Moore DD. Resistance of SHP-null mice to bile acid-induced liver damage. J Biol Chem 278: 4447544481, 2003.[Abstract/Free Full Text]
- Wang L, Lee YK, Bundman D, Han Y, Thevananther S, Kim CS, Chua SS, Wei P, Heyman RA, Karin M, and Moore DD. Redundant pathways for negative feedback regulation of bile acid production. Dev Cell 2: 721731, 2002.[CrossRef][ISI][Medline]
- Yang Y, Zhang M, Eggertsen G, and Chiang JY. On the mechanism of bile acid inhibition of rat sterol 12
-hydroxylase gene (CYP8B1) transcription: roles of alpha-fetoprotein transcription factor and hepatocyte nuclear factor 4
. Biochim Biophys Acta 1583: 6373, 2002.[ISI][Medline]
- Zhang M and Chiang JY. Transcriptional regulation of the human sterol 12
-hydroxylase gene (CYP8B1): roles of hepatocyte nuclear factor 4
(HNF4
) in mediating bile acid repression. J Biol Chem 276: 4169041699, 2001.[Abstract/Free Full Text]
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