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Hematopoietically Expressed Homeobox Is a Target Gene of Farnesoid X Receptor in Chenodeoxycholic Acid–Induced Liver Hypertrophy

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Hematopoietically Expressed Homeobox Is a Target Gene of Farnesoid X Receptor in Chenodeoxycholic Acid–Induced Liver Hypertrophy Hematopoietically Expressed Homeobox Is a Target Gene of Farnesoid X Receptor in Chenodeoxycholic Acid–Induced Liver Hypertrophy Xiangbin Xing,1,2* Elke Burgermeister,1* Fabian Geisler,1 Henrik Einwachter,¨ 1 Lian Fan,1,2 Michaela Hiber,1 Sandra Rauser,3 Axel Walch,3 Christoph Rocken,¨ 4 Martin Ebeling,5 Matthew B. Wright,5 Roland M. Schmid,1 and Matthias P.A. Ebert1 Farnesoid X receptor (FXR/Fxr) is a bile acid–regulated nuclear receptor that promotes hepatic bile acid metabolism, detoxification, and liver regeneration. However, the adap- tive pathways under conditions of bile acid stress are not fully elucidated. We found that wild-type but not Fxr knockout mice on diets enriched with chenodeoxycholic acid (CDCA) increase their liver/body weight ratios by 50% due to hepatocellular hypertro- phy. Microarray analysis identified Hex (Hematopoietically expressed homeobox), a central transcription factor in vertebrate embryogenesis and liver development, as a novel CDCA- and Fxr-regulated gene. HEX/Hex was also regulated by FXR/Fxr and CDCA in primary mouse hepatocytes and human HepG2 cells. Comparative genomic analysis identified a conserved inverted repeat-1–like DNA sequence within a 300 base pair enhancer element of intron-1 in the human and mouse HEX/Hex gene. A combi- nation of chromatin immunoprecipitation, electromobility shift assay, and transcrip- tional reporter assays demonstrated that FXR/Fxr binds to this element and mediates HEX/Hex transcriptional activation. Conclusion: HEX/Hex is a novel bile acid–induced FXR/Fxr target gene during adaptation of hepatocytes to chronic bile acid exposure. (HEPATOLOGY 2009;49:979-988.) iver enlargement (hepatomegaly) is an adaptive re- response. The induction of cytochrome p450 (CYP/Cyp) sponse to prevent toxicity to hepatocytes by en- enzymes and membrane transporters, as well as the Ldobiotics1 or xenobiotics.2 Chronic exposure to growth, differentiation, and cell volume of hepatocytes is endogenous bile acids, as in patients with and rodent regulated by members of the nuclear receptor superfamily models of cholestatic liver diseases,3,4 also activates this such as farnesoid X receptor (FXR/Fxr).1,3 FXR/Fxr binds DNA as a heterodimer with retinoid X receptor and is activated by chenodeoxycholic acid (CDCA).5 FXR/Fxr Abbreviations: CDCA, chenodeoxycholic acid; CYP7A1/Cyp7a1, cholesterol- is a key player in the control of bile acid de novo synthesis, 7alpha-hydroxylase; FXR/Fxr, farnesoid X receptor; HEX/Hex, hematopoietically expressed homeobox; IR-1, inverted repeat-1; SHP/Shp, small heterodimer partner. excretion, and enterohepatic reabsorption. In rodents, a From the 1Department of Medicine II, Klinikum rechts der Isar, Technical high-fat diet, bile acids, and Fxr were linked to liver in- University Munich, Germany; 2 Department of Gastroenterology, The First Affili- jury, inflammation, and fibrosis.6,7 In contrast, studies in 3 ated Hospital of Sun Yat-sen University, Guangzhou, PR China; Institute of Fxr-deficient mice suggest a beneficial role of Fxr in pre- Pathology, Helmholtz Forschungszentrum Mu¨nchen, Neuherberg and 4 Institute of 8 Pathology, Charite, Berlin, Germany; 5 F. Hoffmann-La Roche, Basel, Switzer- vention of cholestasis, in liver regeneration upon partial land. hepatectomy,9 in antimicrobial defense within the gastro- Received May 14, 2008; accepted October 20, 2008. intestinal tract,10 and against tumor formation in the liv- *These authors contributed equally to this study. 11 Address reprints requests to: Prof. Matthias P.A. Ebert, M.D., Department of Med- er. Thus, FXR/Fxr is expected to be a protective and icine II, Klinikum rechts der Isar, Technical University of Mu¨nchen, Ismaningerstraße trophic factor beyond its classical metabolic functions. ϩ 22, D-81675 Mu¨nchen, Germany. E-mail: [email protected]; fax: 49-89- However, the target genes that underlie these functions 4140-2259. Potential conflict of interest: Nothing to report. are still under investigation. We show here that Hex (he- Copyright © 2009 by the American Association for the Study of Liver Diseases. matopoietically expressed homeobox), a key transcription Published online in Wiley InterScience (www.interscience.wiley.com). factor in vertebrate liver development,12 is a novel Fxr- DOI 10.1002/hep.22712 Additional Supporting Information may be found in the online version of this regulated gene that is induced during the liver response to article. CDCA in C57BL/6N mice. 979 980 XING ET AL. HEPATOLOGY, March 2009 Table 1. Sequence Alignment of Predicted* FXR/Fxr-Binding Elements in Intron 1 of Genomic HEX/Hex Loci Element Sequence M00964 RRGGTYA N TRNM (PXR, CAR, LXR, FXR) M00767 GGGTBA A TRACCY (FXR inverted repeat-1) SHP Human GAGTTA A TGACCT (FXR inverted repeat-1) HEX Human CTGTATGAACGGAAAGGGTCA G GCTCTTT-CACTGCACAAGCCTGTTGAA Hex Dog CTGTATGAACGGAAAGGGTCA G GCTCTTT-CACTGCACAAGCTTGTTGAA Hex Cow CTGTATGAACGGAAAGGGTCA G GCTCTTT-CACTGCACAAGCTTGTTGAA Hex Mouse CTGTATGAACGGAAAGGGTCA G GCTCCTT-CACTGCACAAGCTTGTTGAA Hex Rat CTGTATGAACGGAAAGGGTCA G GCTCCTT-CACTGCACAAGCTTGTTGAA Hex opossum CTGTATGAACGGAAAGGGTCA G GCTCTTTTCGCTGCACAAACTTGTTGAA *Based on MLAGAN/TransFac 8.4 matrices (M00964 and M00767). Materials and Methods lands). The 930-bp human HEX proximal promoter (AL590080 [version .25] 24.700 to 25.630 bp) was Animals. Female wild-type (C57BL/6N; Charles cloned into pGL3-basic-luciferase (Promega GmbH, River, Wilmington, MA) and Fxr knockout (Fxr-KO) Mannheim, Germany). The 1428-bp full-length human (B6; 129XFVB-Nr1h4tm1Gonz/J; mixed C57BL/6N back- FXR cDNA (476 amino acids [aa], alpha 1 splice variant ground; Jackson Laboratory, Bar Harbor, ME) mice (4 containing the four-aa insertion MYTG, NM_005123)16,17 weeks, 14-19 g, n ϭ 5 per group) were fed a chow diet was inserted into pTarget (Promega). Transient transfection (Altromin, Lage, Germany) with or without 1% (wt/wt) and luciferase assays were performed as described.14 The CDCA (Chemos GmbH, Regenstauf, Germany). Animal FXR-RE electrophoretic mobility shift assay (EMSA) oligo- studies were conducted in agreement with ethical guide- nucleotide (Supporting Table 2) was generated based on the lines of the Technical University of Munich and approved consensus IR-1 DNA sequence AGG TCA t TGA CCT.18 by the government of Bavaria, Munich, Germany. Immunohistochemistry. Immunohistochemistry (IHC) Reagents. Chemicals were from Merck (Darmstadt, and hematoxylin&eosin staining was performed as de- Germany) or Sigma (Taufkirchen, Germany). Antibodies scribed.19 were FXR/Fxr (sc-13063; Santa Cruz Biotechnology, Reverse Transcription PCR and Quantitative PCR. Santa Cruz, CA); FXR/Fxr (A9033A, R&D Systems, Polymerase chain reactions (PCR) were performed as de- Wiesbaden-Nordenstadt, Germany); HEX/Hex (H-4913; scribed.19 Sigma), lamin A/C (sc-20681; Santa Cruz Biotechnology), DNA Microarray. Total RNA (1 ␮g) from wild-type ␤-actin (AC-74; Sigma), acetyl-histone H3 (06-599; Up- mice on control or CDCA diet (7 days) was subjected to state, Millipore GmbH, Schwalbach, Germany), cyclin D1 One-Cycle cRNA labeling (Affymetrix, Wycombe, UK) (SP4), Ki-67 (SP6) (both from DCS GmbH, Hamburg, and hybridized to a Mouse Genome 430A 2.0 Array (Af- Germany), and bromodeoxyuridine (BrdU; Serotec, Ra- fymetrix). GO-groups were identified by Gene Set En- leigh, NC). richment Analysis (GSEA) (http://www.broad.mit.edu/ Cell Culture. Human embryonic kidney HEK293 gsea) (Supporting Table 3). and hepatoma HepG2 cells (both from the American Comparative Genomics. Homologous genomic loci Type Culture Collection, Manassas, VA) and human (http://genome.ucsc.edu) were identified with BLAST Huh7 cells (JCRB Cell Bank, Japan) were maintained as (Basic Local Alignment Search Tool) and aligned using recommended by the suppliers. Primary mouse hepato- MLAGAN. Binding sites were predicted using matrices cytes were isolated as described.13 from TransFac 8.4 and processed following Rahmann et Oligonucleotides and Plasmids. Reporter plasmids al.20 (Table 1). were based on pTK-luciferase as described.14 A consensus Chromatin Immunoprecipitation. Chromatin Im- IR-1 FXR-responsive element (FXR-RE) GGG ACA t munoprecipitation (ChIP; Upstate, Millipore GmbH) TGA TCC15 from the human bile salt export pump was performed as described.14 (BSEP) Ϫ63/Ϫ50 base pair (bp) promoter sequence Electrophoretic Mobility Shift Assay. Cell extrac- (NT_005403) and a 300-bp fragment of the human tion and western blotting were performed as described.14 HEX intron 1 (AL590080 [version .25] 26.389 to 26.688 Digoxigenin Gel Shift kit (Roche Diagnostics GmbH, bp) (Supporting Table 1) were cloned into pTK-lucif- Mannheim, Germany) and LightShift௡ Chemilumines- erase. Deletion of the IR-1 GGG TCA g GCT CTT in cent EMSA kit (Pierce Biotechnology, Rockford, IL) were HEX intron1–pTK-luciferase was performed by Quick- used as recommended by the manufacturers. change Mutagenesis (Stratagene, Amsterdam, Nether- Statistics. Results are means Ϯ standard error (SE) HEPATOLOGY, Vol. 49, No. 3, 2009 XING ET AL. 981 from five individual animals per group or at least three independent cell experiments. P values were calculated using one-way analysis of variance. Data were analyzed by SPSS 13.0 and Graphpad Prism (Version 4.0). Results Fxr Is Required for Induction of Liver Hypertrophy by CDCA. To stimulate chronic bile acid exposure in mouse liver, C57BL/6N wild-type and Fxr-KO mice were fed a chow diet enriched with 1% (wt/wt) CDCA for 8 weeks. CDCA increased the liver/body weight ratio by approximately 50% (*P Ͻ 0.05) in wild-type
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