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• W2076109204 abstract "Background & Aims: Inhibition of cholesterol 7α-hydroxylase (CYP7A1) by bile acids and inflammatory cytokines provides an important mechanism to protect hepatocytes from bile acid toxicity during cholestasis. Transforming growth factor β1 (TGFβ1) released by hepatic stellate cells during chronic liver injury plays a critical role in liver inflammation and fibrogenesis. The objective of this study is to investigate the role of TGFβ1 in hepatic bile acid synthesis. Methods: mRNA expressions in primary human hepatocytes and HepG2 cells were measured by quantitative real-time polymerase chain reaction. Reporter assay, glutathione-S-transferase pull-down assay, adenovirus-mediated gene transduction, and chromatin immunoprecipitation assay were used to study the mechanism of TGFβ1 regulation of CYP7A1 gene transcription. Results: TGFβ1 inhibited the mRNA expression of CYP7A1 and bile acid synthesis in HepG2 cells and primary human hepatocytes. Mothers against decapentaplegic homolog (Smad3) inhibited both CYP7A1 promoter activity and mRNA expression by inhibiting DNA-binding activity of hepatocyte nuclear factor 4α (HNF4alpha). The histone deacetylase (HDAC) inhibitor Tricostatin A partially blocked the TGFβ1 inhibition of CYP7A1 mRNA expression, whereas TGFβ1 decreased histone 3 acetylation in the CYP7A1 chromatin. TGFβ1 treatment and adenovirus Smad3 reduced HNF4α binding but increased the recruitment of Smad3, HDAC1, and a repressor mSin3A to the CYP7A1 chromatin. Conclusions: This study provides the first evidence that TGFβ1 represses CYP7A1 gene transcription in human hepatocytes by a mechanism involving Smad3-dependent inhibition of HNF4α and HDAC remodeling of CYP7A1 chromatin. The TGFβ1/Smad3 signaling may reduce bile acid synthesis in the liver and prevent hepatocyte injury in cholestatic liver disease. Background & Aims: Inhibition of cholesterol 7α-hydroxylase (CYP7A1) by bile acids and inflammatory cytokines provides an important mechanism to protect hepatocytes from bile acid toxicity during cholestasis. Transforming growth factor β1 (TGFβ1) released by hepatic stellate cells during chronic liver injury plays a critical role in liver inflammation and fibrogenesis. The objective of this study is to investigate the role of TGFβ1 in hepatic bile acid synthesis. Methods: mRNA expressions in primary human hepatocytes and HepG2 cells were measured by quantitative real-time polymerase chain reaction. Reporter assay, glutathione-S-transferase pull-down assay, adenovirus-mediated gene transduction, and chromatin immunoprecipitation assay were used to study the mechanism of TGFβ1 regulation of CYP7A1 gene transcription. Results: TGFβ1 inhibited the mRNA expression of CYP7A1 and bile acid synthesis in HepG2 cells and primary human hepatocytes. Mothers against decapentaplegic homolog (Smad3) inhibited both CYP7A1 promoter activity and mRNA expression by inhibiting DNA-binding activity of hepatocyte nuclear factor 4α (HNF4alpha). The histone deacetylase (HDAC) inhibitor Tricostatin A partially blocked the TGFβ1 inhibition of CYP7A1 mRNA expression, whereas TGFβ1 decreased histone 3 acetylation in the CYP7A1 chromatin. TGFβ1 treatment and adenovirus Smad3 reduced HNF4α binding but increased the recruitment of Smad3, HDAC1, and a repressor mSin3A to the CYP7A1 chromatin. Conclusions: This study provides the first evidence that TGFβ1 represses CYP7A1 gene transcription in human hepatocytes by a mechanism involving Smad3-dependent inhibition of HNF4α and HDAC remodeling of CYP7A1 chromatin. The TGFβ1/Smad3 signaling may reduce bile acid synthesis in the liver and prevent hepatocyte injury in cholestatic liver disease. Bile acids are synthesized from cholesterol exclusively in the liver.1Russell D.W. Setchell K.D. Bile acid biosynthesis.Biochemistry. 1992; 31: 4737-4749Crossref PubMed Scopus (646) Google Scholar, 2Chiang J.Y. Regulation of bile acid synthesis.Front Biosci. 1998; 3: D176-D193PubMed Google Scholar During postprandial state, bile acids are released from gallbladder into the small intestine, where they facilitate the absorption of dietary lipids, fat-soluble vitamins, and steroids. Bile acids are quantitatively reabsorbed in the ileum and transported back to the liver through portal blood circulation to inhibit hepatic bile acid synthesis. Bile acid feedback regulation provides an important mechanism to regulate bile acid homeostasis and prevent hepatic bile acid toxicity during cholestasis. Bile acids inhibit the transcription of the gene encoding cholesterol 7α-hydroxylase (CYP7A1), the rate-limiting enzyme in the classic bile acid biosynthetic pathway. Bile acids activate a bile acid receptor, farnesoid X receptor (NR1H4), which induces an atypical nuclear receptor, small heterodimer partner (NR0B2) to repress CYP7A1 gene transcription.3Goodwin B. Jones S.A. Price R.R. et al.A regulatory cascade of the nuclear receptors FXR, SHP-1, and LRH-1 represses bile acid biosynthesis.Mol Cell. 2000; 6: 517-526Abstract Full Text Full Text PDF PubMed Scopus (1453) Google Scholar Bile acid–activated farnesoid X receptor also induces intestinal expression of fibroblast growth factor 15 (FGF15), which may be transported to the liver to inhibit CYP7A1 gene expression.4Inagaki T. Choi M. Moschetta A. et al.Fibroblast growth factor 15 functions as an enterohepatic signal to regulate bile acid homeostasis.Cell Metab. 2005; 2: 217-225Abstract Full Text Full Text PDF PubMed Scopus (1260) Google Scholar, 5Holt J.A. Luo G. Billin A.N. et al.Definition of a novel growth factor–dependent signal cascade for the suppression of bile acid biosynthesis.Genes Dev. 2003; 17: 1581-1591Crossref PubMed Scopus (520) Google Scholar During cholestasis, high levels of bile acids cause liver inflammation and secretion of a broad array of cytokines from Kupffer cells. Bile acids induce proinflammatory cytokines such as tumor necrosis factor α and interleukin 1β, which have been shown to repress CYP7A1 gene expression via the JNK/cJun pathway.6De Fabiani E. Mitro N. Anzulovich A.C. et al.The negative effects of bile acids and tumor necrosis factor–alpha on the transcription of cholesterol 7alpha-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. 2001; 276: 30708-30716Crossref PubMed Scopus (156) Google Scholar, 7Gupta S. Stravitz R.T. Dent P. et al.Down-regulation of cholesterol 7alpha-hydroxylase (CYP7A1) gene expression by bile acids in primary rat hepatocytes is mediated by the c-Jun N-terminal kinase pathway.J Biol Chem. 2001; 276: 15816-15822Crossref PubMed Scopus (271) Google Scholar, 8Li T. Jahan A. Chiang J.Y. Bile acids and cytokines inhibit the human cholesterol 7 alpha-hydroxylase gene via the JNK/c-jun pathway in human liver cells.Hepatology. 2006; 43: 1202-1210Crossref PubMed Scopus (110) Google Scholar It is well known that inflammation responses following parenchyma cell injuries activate quiescent hepatic stellate cells (HSCs), which undergo transdifferentiation into myofibroblasts that become capable of expressing a broad array of cytokines and most components of extracellular matrix, leading to inflammation, extracellular matrix accumulation, and liver fibrosis.9Gressner A.M. Weiskirchen R. Modern pathogenetic concepts of liver fibrosis suggest stellate cells and TGF-beta as major players and therapeutic targets.J Cell Mol Med. 2006; 10: 76-99Crossref PubMed Scopus (676) Google Scholar The transforming growth factor β1(TGFβ1), an autocrine/paracrine cytokine secreted by activated HSCs, plays a major role in the activation and proliferation of HSCs and stimulation of extracellular matrix expression. TGFβ1 is considered the most effective profibrogenic mediator during liver fibrogenesis. TGFβ1 signals through the transmembrane cell surface TGFβ receptor type I (TβRI) and type II (TβRII) that possess intracellular serine/threonine kinase activity. Following TGFβ1 binding to TβRII, TβRII recruits and phosphorylates TβRI at a glycine/serine-rich region (GS box) and becomes activated. The downstream events of TβRI are mediated by the transcription factors Smad2 and Smad3. In a typical TGFβ1 signaling, TGFβ1-activated TβRI recruits and phosphorylates Smad2 and Smad3 at a conserved C-terminal SSXS motif. Phosphorylated Smad2 or Smad3 either homodimerizes or heterodimerizes with Smad4, translocates to the nucleus to bind to DNA and other transcription factors, and regulates gene transcription.10Heldin C.H. Miyazono K. ten Dijke P. TGF-beta signaling from cell membrane to nucleus through SMAD proteins.Nature. 1997; 390: 465-471Crossref PubMed Scopus (3301) Google Scholar, 11Derynck R. Zhang Y.E. Smad-dependent and Smad-independent pathways in TGF-beta family signaling.Nature. 2003; 425: 577-584Crossref PubMed Scopus (4156) Google Scholar Smad proteins can regulate gene transcription both positively and negatively in a gene- and tissue-specific manner. The primary structure of Smad3 consists of two conserved domains: N-terminal MH1 and C-terminal MH2 linked by a proline-rich linker region. The MH1 domain of Smad3 recognizes an 8 base-pair DNA sequence GTCTGTCT in the target genes. In contrast, Smad2 does not bind directly to DNA because the amino acids that are responsible for DNA binding are displaced in Smad2. The MH2 domain of Smad2 and Smad3 is responsible for their interaction with many transcription factors and coregulators. When binding to DNA, Smad3 has been shown to recruit coactivator CBP/P300 to activate gene transcriptions.12Janknecht R. Wells N.J. Hunter T. TGF-Beta–stimulated cooperation of smad proteins with the coactivators CBP/p300.Genes Dev. 1998; 12: 2114-2119Crossref PubMed Scopus (432) Google Scholar Smad3, but not Smad2, also interacts with transcriptional factors Sp1 and nuclear receptor HNF4α and synergistically regulates gene transcription in HepG2 cells.13Moustakas A. Kardassis D. Regulation of the human p21/WAF1/Cip1 promoter in hepatic cells by functional interactions between Sp1 and Smad family members.Proc Natl Acad Sci U S A. 1998; 95: 6733-6738Crossref PubMed Scopus (319) Google Scholar, 14Chou W.C. Prokova V. Shiraishi K. et al.Mechanism of a transcriptional cross talk between transforming growth factor-beta–regulated Smad3 and Smad4 proteins and orphan nuclear receptor hepatocyte nuclear factor-4.Mol Biol Cell. 2003; 14: 1279-1294Crossref PubMed Scopus (41) Google Scholar However, much less is known about TGFβ1/Smad-mediated inhibition of gene transcription. It has been shown that Smad3 can interact with transcription factor myocyte enhancer factor 2 in myoblast and block its interaction with coactivator glucocorticoid receptor interacting protein.15Liu D. Kang J.S. Derynck R. TGF-beta–activated Smad3 represses MEF2-dependent transcription in myogenic differentiation.Embo J. 2004; 23: 1557-1566Crossref PubMed Scopus (110) Google Scholar Several studies have suggested that Smad2 and Smad3 may inhibit gene transcription by recruiting histone deacetylase (HDACs), either directly or indirectly through interacting with corepressor complex.16Luo K. Stroschein S.L. Wang W. et al.The Ski oncoprotein interacts with the Smad proteins to repress TGF-beta signaling.Genes Dev. 1999; 13: 2196-2206Crossref PubMed Scopus (387) Google Scholar, 17Stroschein S.L. Wang W. Zhou S. et al.Negative feedback regulation of TGF-beta signaling by the SnoN oncoprotein.Science. 1999; 286: 771-774Crossref PubMed Scopus (433) Google Scholar, 18Kang J.S. Alliston T. Delston R. et al.Repression of Runx2 function by TGF-beta through recruitment of class II histone deacetylases by Smad3.Embo J. 2005; 24: 2543-2555Crossref PubMed Scopus (271) Google Scholar Although TGFβ1 is an important cytokine mediator of various hepatic injuries and liver fibrosis, its role in modulating bile acid homeostasis has not been investigated. In this study, we investigated the role of TGFβ1 in transcriptional regulation of human CYP7A1 gene in primary human hepatocytes and HepG2 cells. We showed that TGFβ1 strongly and specifically repressed CYP7A1 gene transcription through Smad3-dependent inhibition of HNF4α. Our study has identified a novel pathway that may prevent hepatic bile acid toxicity during cholestatic liver disease. The human hepatoblastoma cell line, HepG2, was purchased from American Type Culture Collection (Manassas, VA). The cells were cultured in Dulbecco’s modified Eagle medium and F-12 (Sigma, St. Louis, MO) supplemented with 100 U/mL penicillin G/streptomycin sulfate (Mediatech, Herndon, VA) and 10% (vol/vol) heat inactivated fetal bovine serum (Irvine Scientific, Santa Ana, CA). Primary human hepatocytes were isolated from human donors and were obtained through the Liver Tissue Procurement and Distribution System of National Institutes of Health (S. Strom, University of Pittsburgh, Pittsburgh, PA). Cells were maintained in Hepatocyte Maintenance Medium supplemented with 10−7 mol/L insulin and dexamethasone (Clonetics, Cambrex Bioscience, Walkersville, MD). Human CYP7A1/Luc reporters (ph-1877, ph-371, ph-150, ph-80, and HNF site mutant reporter, mHNF4-ph-1887Luc) were constructed as previously described.19Crestani M. Stroup D. Chiang J.Y. Hormonal regulation of the cholesterol 7 alpha-hydroxylase gene (CYP7).J Lipid Res. 1995; 36: 2419-2432Abstract Full Text PDF PubMed Google Scholar, 20Wang D.P. Stroup D. Marrapodi M. et al.Transcriptional regulation of the human cholesterol 7α-hydroxylase gene (CYP7A) in HepG2 cells.J Lipid Res. 1996; 37: 1831-1841Abstract Full Text PDF PubMed Google Scholar Expression plasmids pFLAG-CMV2-Smad2, pFLAG-CMV2-Smad3, and pFLAG-CMV2-Smad4 expressing N-terminal Flag-tagged proteins were kindly provided by Dr David Jones (University of Utah, Salt Lake City, UT). The cDNA fragments for Smad2, Smad3, and Smad4 were digested by EcoRI and XbaI and subsequently inserted into pcDNA3.1. The ATG start codon was then introduced into the pcDNA3.1-Smad2, 3, and 4 plasmids by mutagenesis. Expression plasmid for human PGC-1α (pcDNA3/HA-PGC-1α) was obtained from Dr A. Kralli (The Scripps Research Institute, La Jolla, CA). The construction of the expression plasmid for HNF4α (pCMV-HNF4α) was previously described.21Crestani M. Sadeghpour A. Stroup D. et al.Transcriptional activation of the cholesterol 7alpha-hydroxylase gene (CYP7A) by nuclear hormone receptors.J Lipid Res. 1998; 39: 2192-2200Abstract Full Text Full Text PDF PubMed Google Scholar The β-galactosidase expression plasmid (pCMV-β), and the mammalian expression vector pcDNA3.1 were obtained from Clonetech (Palo Alto, CA). The reporter vector pGL3-Basic was purchased from Promega (Madison, WI). The heterologous promoter reporter 5XUAS-TK-Luc was kindly provided by Dr A. Takeshita (Toranomon Hospital, Tokyo, Japan) and described elsewhere.22Li T. Chiang J.Y. Mechanism of rifampicin and pregnane X receptor inhibition of human cholesterol 7 alpha-hydroxylase gene transcription.Am J Physiol Gastrointest Liver Physiol. 2005; 288: G74-G84Crossref PubMed Scopus (175) Google Scholar Gal4-HNF4α fusion plasmid pBx-HNF4-LBD was obtained from Dr I. Talianidis (Institute of Molecular Biology and Biotechnology Foundation for Research and Technology, Hellas, Herakleion Crete, Greece). The 4×HNF4/TK/Luc reporter containing 4 copies of HNF4α binding site upstream of the thymidine kinase promoter (TK) was provided by Dr H. S. Choi (Chungnam National University, Korea). Luciferase reporters and expression plasmids were transfected into HepG2 cells using Lipofectamin 2000 reagent (Life Technologies Inc, Gaithersburg, MD) following manufacturer’s instructions. Luciferase reporter activities were assayed and expressed as relative luciferase units divided by β-galactosidase activity as described previously.22Li T. Chiang J.Y. Mechanism of rifampicin and pregnane X receptor inhibition of human cholesterol 7 alpha-hydroxylase gene transcription.Am J Physiol Gastrointest Liver Physiol. 2005; 288: G74-G84Crossref PubMed Scopus (175) Google Scholar Assays were performed in triplicates and expressed as mean ± SD. HepG2 cells and primary human hepatocytes were cultured in serum-free and insulin-free media for 24 hours before addition of treatment. RNA isolation, reverse transcription reactions, and real-time PCR were performed as described previously.23Song K.H. Chiang J.Y. Glucagon and cAMP inhibit cholesterol 7alpha-hydroxylase (CYP7A1) gene expression in human hepatocytes: discordant regulation of bile acid synthesis and gluconeogenesis.Hepatology. 2006; 43: 117-125Crossref PubMed Scopus (47) Google Scholar All primers/probes used were TaqMan Gene Expression Assays purchased from Applied Biosystems. Amplification of Ubiquitin C was used in the same reactions of all samples as an internal control. Relative mRNA expression was quantified using the comparative CT (Ct) method and expressed as 2−ΔΔCt. HepG2 cells were cultured in a 100-mm tissue culture dish. Cell nuclei were used for chromatin immunoprecipitation (ChIP) assays as described previously.22Li T. Chiang J.Y. Mechanism of rifampicin and pregnane X receptor inhibition of human cholesterol 7 alpha-hydroxylase gene transcription.Am J Physiol Gastrointest Liver Physiol. 2005; 288: G74-G84Crossref PubMed Scopus (175) Google Scholar Antibodies against Acetyl-histone3 (Upstate Biotech, Charlottesville, VA), HNF4α, Smad2/3, mSin3A, PGC-1α (Santa Cruz Biotechnology, Santa Cruz, CA) and HDAC1 (Abcam, Cambridge, MA) were used to immunoprecipitate chromatin. For ChIP assays with anti-acetyl-histone3, PCR primers were designed to detect a 391-bp fragment (−432/−41) on CYP7A1 promoter, a 394-bp fragment from CYP7A1 intron 2 (+2485/+2879), a 268-bp DNA fragment (−291/−23) on steroid 27-hydroxylase (CYP27A1) promoter, and a 376-bp fragment from CYP27A1 intron 1 region (+4029/+4405). For ChIP assays with other antibodies, absolute quantification method of real-time PCR was used to quantify ChIP assay results. Taqman primers/probe sets (Supplemental Figure 1; see supplemental material online at www.gastrojournal.org) were designed to detect CYP7A1 promoter region containing a bile acid response element (HNF4α and LRH binding site, −180 to −111) and an intron 5 region by real-time PCR. Standard curve: Ct versus Log2 (ng of chromatin) for both promoter and intron 5 primers/probe sets were made with sonicated and purified chromatin from the same ChIP assay sample. The amount of immunoprecipitated CYP7A1 promoter chromatin was determined as nanograms of chromatin from standard curve. The nonspecific background, which was reflected by the amount of intron 5 chromatin detected in the same IP sample, was subtracted from the precipitated CYP7A1 chromatin. The relative binding strength was expressed in arbitrary unit with control set as “1.” GST or GST full-length human HNF4α fusion protein was expressed in E coli BL21 cells. Smad2, Smad3, and Smad4 were in vitro translated and radiolabeled with [35S]-methionine. GST pull-down assays were performed as described previously.24Li T. Chiang J.Y. Rifampicin induction of CYP3A4 requires PXR crosstalk with HNF4{alpha} and co-activators, and suppression of SHP gene expression.Drug Metab Dispos. 2006; Google Scholar Total cell lysates or nuclei fractions were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Antibodies against Smad2/3, phospho-Smad2/3, Actin, and lamin B (Santa Cruz Biotechnology, CA) were used for immunoblotting and detected by ECL Western blotting detection kit (Amersham Biosciences, UK). Adeno-EGFP was obtained from Dr Li Wang (University of Kansas Medical Center, Kansas, KS), Adeno-Smad2 and Smad3 were purchased from Vector Biolabs (Philadelphia, PA). Recombinant adenoviruses were amplified in HEK293A cells and purified with Adeno-X Virus mini purification kit (BD Biosciences, San Jose, CA). Virus titer was determined by adeno-X rapid titer kit (BD Biosciences). HepG2 cells were plated on chamber slides and treated with TGFβ1 (0.1 nmol/L) for 1 hour. Cells were fixed with 4% formaldehyde and permeabilized with 0.1% Triton X100. Anti-Smad2/3 first antibody and Alexa Fluor 488 conjugated secondary antibody (Molecular Probes, Carlsbad, CA) were used for Smad2/3 detection under a confocal microscopy. Cell nuclei were counterstained with TO-PRO-3 (Molecular Probes). Nonimmune IgG was used as background control. Total bile acids from cells and medium was extracted with Sep-Pak C18 cartridge and quantified with total bile acid colorimetric assay kit (Bio-Quant, San Diego, CA) following the manufacturer’s instruction. All results were expressed as mean ± SD. Real-time PCR data of primary human hepatocytes were analyzed with paired t test. Real-time PCR of HepG2 cells and reporter assays were performed in triplicates and data analyzed with Student’s t test. P values of < .05 were considered as statistically significant difference between treated and untreated control. In healthy human, average serum TGFβ1 concentration is approximately 1 nmol/L (between approximately 0.1 nmol/L and 2 nmol/L).25Vesely D. Astl J. Lastuvka P. et al.Serum levels of IGF-I, HGF, TGFbeta1, bFGF and VEGF in thyroid gland tumors.Physiol Res. 2004; 53: 83-89PubMed Google Scholar We first examined the time-dependent effect of TGFβ1 on human CYP7A1 mRNA expression in HepG2. TGFβ1 at 0.1 nmol/L strongly and rapidly inhibited CYP7A1 mRNA with maximum inhibition reached at 2 hours (Figure 1A). In contrast, TGFβ1 did not inhibit the mRNA expression of sterol 12α-hydroxylase (CYP8B1) and sterol 27-hydroxylase (CYP27A1) (Figure 1A). TGFβ1 treatment for 2 hours also strongly inhibited CYP7A1 mRNA in a dose-dependent manner (Figure 1B). TGFβ1 (0.1 nmol/L) strongly inhibited CYP7A1 mRNA in 7 donor hepatocytes, while donors had only weak to no inhibition in 3 donors (Figure 1C). TGFβ1 (0.1 nmol/L for 24 hours) inhibited total bile acid synthesis in HepG2 cells and primary hepatocytes by 30% to 50% (Figure 1D). Under these experimental conditions TGFβ1 had no cytotoxicity to HepG2 and primary human hepatocytes (Supplemental Figure 2; see supplemental material online at www.gastrojournal.org). Taken together, these results suggest that TGFβ1 inhibits bile acid synthesis and CYP7A1 gene expression in hepatocytes, and TGFβ1 inhibition of CYP7A1 in human livers varies significantly among individuals. Reporter assays with CYP7A1 promoter deletion constructs mapped the TGFβ1 responsive element between the −150 and −80 region, which contains an HNF4α binding site (Supplemental Figure 3A; see supplemental material online at www.gastrojournal.org). HNF4α plays a critical role in regulation of human CYP7A1 gene transcription. Mutation of the HNF4α binding site (mHNF4-ph1887-luc) dramatically reduced the reporter activity and abolished the inhibitory effect of TGFβ1 (Supplemental Figure 3B; see supplemental material online at www.gastrojournal.org). These results indicate that TGFβ1 may inhibit CYP7A1 by targeting to HNF4α. It is known that TGFβ1 exerts most of its biological effects through activation of Smad2 and Smad3.10Heldin C.H. Miyazono K. ten Dijke P. TGF-beta signaling from cell membrane to nucleus through SMAD proteins.Nature. 1997; 390: 465-471Crossref PubMed Scopus (3301) Google Scholar We thus studied the effect of Smad2, Smad3, and Smad4 on the human CYP7A1 reporter activity. Cotransfection of Smad3 alone or in combination with Smad2 and/or Smad4 strongly inhibited the CYP7A1 reporter activity, which was further suppressed by TGFβ1 treatment at 0.1 nmol/L for 6 hours (Figure 2A and B). In contrast, cotransfection of Smad2 or Smad4 alone or in combination failed to inhibit CYP7A1 reporter activity. These results suggest that Smad3, but not Smad2 or Smad4, mediates TGFβ1 inhibition of CYP7A1. Analysis of CYP7A1 promoter sequence did not identify any putative Smad3 binding site. Subsequently, reporter assays using CYP7A1 reporter deletion constructs located the Smad3-responsive region to between −150 and −80 that contains a HNF4α binding site, and mutations of the HNF4α binding site abolished Smad3 inhibition of CYP7A1 reporter activity (Figure 3A and B). It has been reported that Smad3 and Smad4, but not Smad2, interact with HNF4α at both N-terminal AF-1 domain and C-terminal F domain.14Chou W.C. Prokova V. Shiraishi K. et al.Mechanism of a transcriptional cross talk between transforming growth factor-beta–regulated Smad3 and Smad4 proteins and orphan nuclear receptor hepatocyte nuclear factor-4.Mol Biol Cell. 2003; 14: 1279-1294Crossref PubMed Scopus (41) Google Scholar Consistently, our in vitro GST pull-down assay indicated that GST-HNF4α interacted with Smad3 and Smad4 but not with Smad2 (Figure 3C). To study the effect of Smad3 on HNF4α transactivation activity, we performed reporter assay using a heterologous reporter, 4xHNF4α-tk-luc reporter. Cotransfection of Smad3, but not Smad2 or Smad4, strongly inhibited HNF4α stimulation of the 4×HNF4-TK-Luc reporter activity by approximately 80% (Figure 3D). We then did mammalian one-hybrid assay using a Gal4-HNF4-LBD fusion plasmid in which the HNF4α ligand-binding domain (LBD) is fused to the Gal4 DNA binding domain (DBD). Interestingly, cotransfection of Smad3 only inhibited PGC-1α and Gal4-HNF4α-LBD coactivation of the reporter activity by approximately 20% (Figure 3E). Taken together, these results suggest that Smad3 may inhibit HNF4α transactivating activity mainly by interfering with HNF4α DNA binding activity. Reduced coactivator recruitment may also contribute to Smad3-dependent repression of HNF4α.Figure 3Smad3 interacts with HNF4α to inhibit CYP7A1. (A) Human CYP7A1 promoter deletion constructs (0.2 μg) was cotransfected with pcDNA3 or Smad3 (0.1 μg). An asterisk (*) indicates statistically significant difference (P < .05, n = 3), pcDNA3 control plasmid versus Smad3. (B) Human CYP7A1 HNF4α site mutant reporter construct (mHNF4-ph-1887-luc, 0.2 μg) was cotransfected with Smad3 (0.1 μg) into hepG2 cells for reporter assay. (C) glutathione S transferase (GST) pull-down assay. Bacterially expressed GST or GST-HNF4α full-length fusion protein and [S35] labeled Smad2, Smad 3, and Smad 4 were used. (D) A heterologous promoter reporter (0.2 μg) containing 4 HNF4α binding sites was cotransfected with 0.1 μg HNF4α and/or Smad2, Smad3, and/or Smad4 expression plasmids. (E) As indicated, 5 X UAS luciferase reporter (0.2 μg) containing 5 Gal4 binding sites was cotransfected with 0.1 μg Gal4-HNF4-LBD fusion plasmid, PGC-1α and/or Smad2, Smad 3, and Smad 4. An asterisk (*) indicates statistically significant difference (P < .05, n = 3), TGFβ1-treated or plasmid-transfected versus nontreated or empty plasmid-transfected control.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To further confirm the role of Smad3 in CYP7A1 gene repression, we treated HepG2 cells with TGFβ1 for a period of time and monitored Smad3 by Western blot. As shown in Figure 4A, Smad2 and Smad3 protein levels were not significantly altered by TGFβ1. Without TGFβ1 treatment, phosphorylated Smad2 and Smad3 were detected, indicating that Smad2 and Smad3 may have constitutive activity in liver cells. TGFβ1 treatment rapidly increased the phosphorylation of Smad2 and Smad3 in 30 minutes and resulted in a rapid nuclear accumulation of Smad2 and Smad3 in HepG2 cells (Figure 4B). Consistently, immunofluorescent staining in HepG2 cells showed that without TGFβ1, Smad2 and Smad3 are localized in both cytoplasm and nuclei, and TGFβ1 treatment caused translocation of Smad2 and Smad3 exclusively in the nuclei (Supplemental Figure 4; see supplemental material online at www.gastrojournal.org). We next overexpressed Smad3 in HepG2 cells by adenovirus-mediated gene transduction. Western blot analysis showed that overexpression of Smad3 by adenovirus led to a robust Smad3 protein accumulation in the HepG2 cell nuclei (Figure 4C). Smad3 strongly inhibited CYP7A1 mRNA level but not CYP27A1 or CYP8B1 mRNA expression (Figure 4D). In contrast, overexpression of Smad2 did not inhibit CYP7A1 mRNA (Supplemental Figure 5; see supplemental material online at www.gastrojournal.org). These data support a major role of Smad3 in mediating TGFβ1 regulation of CYP7A1. We next tested whether HDAC is involved in TGFβ1 inhibition of CYP7A1. As shown in Figure 5A, when HepG2 cells were pretreated with the HDAC inhibitor tricostatin A (TSA), TGFβ1 (2 hours) inhibition of CYP7A1 mRNA was partially blocked. Similar results were observed when cells were treated with TGFβ1 for 1 hour, 3 hours, or 6 hours (data not shown). GST pull-down assay using bacterially expressed GST-HNF4α and HepG2 cell lysate showed that HNF4α could form a protein complex with Smad3 (Figure 5B). GST pull-down assays also detected weak interaction of HNF4α with a repressor mSin3A and HDAC1 (Figure 5B). These results are consistent with a previous report and suggest that Smad3 can recruit a repressor complex that contains mSin3A and HDACs to regulate gene transcription via chromatin remodeling.26Liberati N.T. Moniwa M. Borton A.J. et al.An essential role for Mad homology domain 1 in the association of Smad3 with histone deacetylase activity*.J Biol Chem. 2001; 276: 22595-22603Crossref PubMed Scopus (33) Google Scholar We then performed ChIP assay in HepG2 cells with an antibody against acetyl-histone 3 to study the effect of TGFβ1 on the acetylation status of histones in CYP7A1 chromatin. Figure 5C shows that TGFβ1 (0.1 nmol/L) treatment for 2 hours and 6 hours significantly decreased the acetylation levels of histone 3 in the CYP7A1 chromatin, consistent with HDAC-mediated chromatin remodeling and gene repression. In contrast, acetylation status of histone 3 on CYP27A1 gene was not changed, consistent with lack of effect of CYP27A1 gene expression by TGFβ1. These results suggest that TGFβ1-activated Smad3 may recruit HDAC activity to deacetylate histones in CYP7A1 chromatin and result in inhibiting CYP7A1 gene expression. We then studied the effect of TGFβ1 on CYP7A1 chromatin structure using ChIP assay coupled with real-time PCR analysis. Figure 6 shows that TGFβ1 treatment for 2 hours decreased the amount of HNF4α and CBP (a histone acetylase) in CYP7A1 chromatin by approximately 50% but increased Smad3, mSin3A, and HDAC1 association with the CYP7A1 chromatin. Figure 7 shows that when Smad3 was overexpressed in HepG2 cells by adenovirus-mediated gene transduction, HNF4α and CBP association with the CYP7A1 chromatin was reduced whereas Smad3, mSin3A, and HDAC1 association with the CYP7A1 chromatin was increased. These data demonstrated that Smad3 might inhibit CYP7A1 by interfering with HNF4α binding to DNA, inhibiting CBP recruitment to chromatin, and enhancing corepressor and HDAC1 recruitment to CYP7A1 chromatin.Figure 7ChIP of effect of Smad3 overexpression on CYP7A1 chromatin structure. HepG2 cells were infected with adenovirus expressing either EGFP or Smad3 at MOI 30 for 36 hours. ChIP assay was performed as described in Materials and Methods.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In this study, we have provided the first evidence that the profibrogenic cytokine TGFβ1 inhibits human CYP7A1 gene transcription. TGFβ1 inhibits CYP7A1 mRNA expression and bile acid synthesis in both primary human hepatocytes and HepG2 cells. Our study indicates that TGFβ1 causes phosphorylation and activation of Smad3, which enters the nucleus to inhibit human CYP7A1 gene transcription through direct interaction with HNF4α. Smad3 may inhibit HNF4α transactivating activity through 2 modes of action: first, it may interact with the N-terminal domain of HNF4α and reduce its DNA binding affinity; second, it may interfere with HNF4α coactivator recruitment through its interaction with the C-terminal domain of HNF4α and recruit corepressor complexes and HDAC to inhibit CYP7A1 gene transcription (Figure 8). It is unclear why CYP7A1 mRNA expressions in primary hepatocytes of certain donors were less responsive to TGFβ1 treatment. It is possible that the endogenous TGFβ1 levels may vary widely among different donor livers because of certain pathological conditions such as fatty liver, fibrosis, inflammation, and proliferation. Under these conditions, the TGFβ1/Smad3 signaling may be already activated or inactivated in some donor hepatocytes. It is known that activated TGFβ1/Smad3 pathway can be inhibited by a feedback mechanism; Smad3 induces Ski and Sno oncoproteins, which interact with Smad3 and inhibit TGFβ1/Smad3 signaling.16Luo K. Stroschein S.L. Wang W. et al.The Ski oncoprotein interacts with the Smad proteins to repress TGF-beta signaling.Genes Dev. 1999; 13: 2196-2206Crossref PubMed Scopus (387) Google Scholar, 17Stroschein S.L. Wang W. Zhou S. et al.Negative feedback regulation of TGF-beta signaling by the SnoN oncoprotein.Science. 1999; 286: 771-774Crossref PubMed Scopus (433) Google Scholar Accumulation of high levels of cholestatic bile acids in the liver causes inflammation and liver injuries. In acute-phase response to injury, hepatocytes and adjacent Kupffer cells secrete high levels of reactive oxygen species and proinflammatory cytokines (tumor necrosis factor α, IL-1α), which inhibit CYP7A1 and bile acid synthesis.8Li T. Jahan A. Chiang J.Y. Bile acids and cytokines inhibit the human cholesterol 7 alpha-hydroxylase gene via the JNK/c-jun pathway in human liver cells.Hepatology. 2006; 43: 1202-1210Crossref PubMed Scopus (110) Google Scholar High levels of TGFβ1 are often found in cholestatic livers. Bile acids at cholestatic levels can directly stimulate HSC proliferation through protein kinase C–dependent induction of the epidermal growth factor receptor.27Svegliati-Baroni G. Ridolfi F. Hannivoort R. et al.Bile acids induce hepatic stellate cell proliferation via activation of the epidermal growth factor receptor.Gastroenterology. 2005; 128: 1042-1055Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar Bile acids induce thrombospondin-1, an activator of latent TGFβ1, in hepatocytes and also contribute to HSC proliferation and transdifferentiation.28Myung S.J. Yoon J.H. Gwak G.Y. et al.Bile acid–mediated thrombospondin-1 induction in hepatocytes leads to transforming growth factor-beta–dependent hepatic stellate cell activation.Biochem Biophys Res Commun. 2007; 353: 1091-1096Crossref PubMed Scopus (18) Google Scholar Thus, bile acids are promoters of liver fibrosis through activation of HSCs. Inhibition of bile acid synthesis is important in reducing both hepatocyte injury and HSC activation in the fibrogenic process. Recently, FGF15 has been identified as a bile acid–induced intestine cytokine that is transported to hepatocyte to inhibit CYP7A1 gene transcription.4Inagaki T. Choi M. Moschetta A. et al.Fibroblast growth factor 15 functions as an enterohepatic signal to regulate bile acid homeostasis.Cell Metab. 2005; 2: 217-225Abstract Full Text Full Text PDF PubMed Scopus (1260) Google Scholar This finding may provide an explanation for the observation that CYP7A1 activity is paradoxically increased in the liver of bile duct ligated rats29Dueland S. Reichen J. Everson G.T. et al.Regulation of cholesterol and bile acid homeostasis in bile-obstructed rats.Biochem J. 1991; 280: 373-377Crossref PubMed Scopus (104) Google Scholar and in human patients with obstructive jaundice.30Bertolotti M. Carulli L. Concari M. et al.Suppression of bile acid synthesis, but not of hepatic cholesterol 7alpha-hydroxylase expression, by obstructive cholestasis in humans.Hepatology. 2001; 34: 234-242Crossref PubMed Scopus (27) Google Scholar However, a more recent study shows slight or no increase of CYP7A1 and strong reduction of CYP8B1 mRNA expression in rat models of intrahepatic and extrahepatic cholestasis.31Liu Y. Binz J. Numerick M.J. et al.Hepatoprotection by the farnesoid X receptor agonist GW4064 in rat models of intra- and extrahepatic cholestasis.J Clin Invest. 2003; 112: 1678-1687Crossref PubMed Scopus (344) Google Scholar Furthermore, FGF19, a human ortholog of FGF15, is strongly induced by bile acids in human primary hepatocytes. It appears that FGF19 inhibits CYP7A1 expression by an autocrine/paracrine mechanism (preliminary results from this laboratory). TFGβ1 signaling is blocked during liver proliferation to allow DNA synthesis.32Taub R. Liver regeneration: from myth to mechanism.Nat Rev Mol Cell Biol. 2004; 5: 836-847Crossref PubMed Scopus (1230) Google Scholar It is possible that the observed increase or no effect of CYP7A1 activity and mRNA expression may be due to liver regeneration response to injury. The complex interplay of cytokines and growth factors induced during liver injury may modulate the rate of bile acid synthesis to maintain lipid homeostasis in the liver. In accordance with this finding, several recent studies have indicated that regulatory crosstalk between hepatocytes and HSCs may exist to control bile acid toxicity and HSC activation in liver. Recently, an antifibrotic role of farnesoid X receptor in inhibiting HSC activation has also been demonstrated in rodent models of liver cirrhosis.33Fiorucci S. Antonelli E. Rizzo G. et al.The nuclear receptor SHP mediates inhibition of hepatic stellate cells by FXR and protects against liver fibrosis.Gastroenterology. 2004; 127: 1497-1512Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar, 34Fiorucci S. Rizzo G. Antonelli E. et al.A farnesoid x receptor–small heterodimer partner regulatory cascade modulates tissue metalloproteinase inhibitor-1 and matrix metalloprotease expression in hepatic stellate cells and promotes resolution of liver fibrosis.J Pharmacol Exp Ther. 2005; 314: 584-595Crossref PubMed Scopus (167) Google Scholar Interestingly, insulin-regulated forkhead transcriptional factor O1 (FoxO1) inhibits the proliferation and transdifferentiation of HSCs and may play an important role in protecting against liver fibrosis.35Adachi M. Osawa Y. Uchinami H. et al.The forkhead transcription factor FoxO1 regulates proliferation and transdifferentiation of hepatic stellate cells.Gastroenterology. 2007; 132: 1434-1446Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar Our recent study shows that FoxO1 inhibits CYP7A1 gene transcription in human hepatocytes and insulin signaling rapidly stimulates CYP7A1 gene expression by inhibiting FoxO1 activity.36Li T. Kong X. Owsley E. et al.Insulin regulation of cholesterol 7alpha-hydroxylase expression in human hepatocytes: roles of forkhead box O1 and sterol regulatory element-binding protein 1c.J Biol Chem. 2006; 281: 28745-28754Crossref PubMed Scopus (68) Google Scholar Therefore, FoxO1 may inhibit bile acid synthesis and HSC activation to protect against liver fibrosis. In summary, this study has revealed a novel role of TGFβ1 in regulation of CYP7A1 gene transcription. The TGFβ1 inhibition of CYP7A1 may provide an important mechanism to control bile acid homeostasis and to prevent hepatic bile acid toxicity during cholestatic liver injuries and liver fibrosis. Download .tif (.07 MB) Help with tif files Supplementary Figure Download .tif (.07 MB) Help with tif files Supplementary Figure Download .tif (.11 MB) Help with tif files Supplementary Figure Download .tif (.37 MB) Help with tif files Supplementary Figure Download .tif (.12 MB) Help with tif files Supplementary Figure" @default.
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• W2076109204 title "A Novel Role of Transforming Growth Factor β1 in Transcriptional Repression of Human Cholesterol 7α-Hydroxylase Gene" @default.
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