Retinoic acid represses CYP7A1 expression in human hepatocytes and HepG2 cells by FXR/RXR-dependent and independent mechanisms
2010; Elsevier BV; Volume: 51; Issue: 8 Linguagem: Inglês
10.1194/jlr.m005546
ISSN1539-7262
AutoresShi‐Ying Cai, Hongwei He, Trọng Tuân Nguyễn, Albert Mennone, James L. Boyer,
Tópico(s)Peroxisome Proliferator-Activated Receptors
ResumoCholesterol 7α-hydroxylase (CYP7A1) plays a key role in maintaining lipid and bile salt homeostasis as it is the rate-limiting enzyme converting cholesterol to bile acids. Deficiency of CYP7A1 leads to hyperlipidemia in man and mouse. Hyperlipidemia is often seen in patients when treated with high-dose retinoic acid (RA), but the molecular mechanisms remain elusive. Our present study revealed that CYP7A1 mRNA expression is greatly repressed by RA in both human hepatocytes and HepG2 cells where increased fibroblast growth factor 19 (FGF19) and small heterodimer partner (SHP) expressions were also observed, suggesting farnesoid X receptor (FXR) and retinoid X receptor (RXR) were activated. Promoter reporter assays demonstrate that all-trans RA (atRA) specifically activated FXR/RXR. However, detailed molecular analyses indicate that this activation is through RXR, whose ligand is 9-cis RA. Knocking down of FXR or RXRα by small interference RNA (siRNA) in human hepatocytes increased CYP7A1 basal expression, but the repressive effect of atRA persisted, suggesting there are also FXR/RXR-independent mechanisms mediating atRA repression of CYP7A1 expression. Chromatin immunoprecipitation (ChIP) assay and cell transfection results indicate that PGC-1α plays a role in the FXR/RXR-independent mechanism. Our findings may provide a potential explanation for hyperlipidemic side effects observed in some patients treated with high-dose RA. Cholesterol 7α-hydroxylase (CYP7A1) plays a key role in maintaining lipid and bile salt homeostasis as it is the rate-limiting enzyme converting cholesterol to bile acids. Deficiency of CYP7A1 leads to hyperlipidemia in man and mouse. Hyperlipidemia is often seen in patients when treated with high-dose retinoic acid (RA), but the molecular mechanisms remain elusive. Our present study revealed that CYP7A1 mRNA expression is greatly repressed by RA in both human hepatocytes and HepG2 cells where increased fibroblast growth factor 19 (FGF19) and small heterodimer partner (SHP) expressions were also observed, suggesting farnesoid X receptor (FXR) and retinoid X receptor (RXR) were activated. Promoter reporter assays demonstrate that all-trans RA (atRA) specifically activated FXR/RXR. However, detailed molecular analyses indicate that this activation is through RXR, whose ligand is 9-cis RA. Knocking down of FXR or RXRα by small interference RNA (siRNA) in human hepatocytes increased CYP7A1 basal expression, but the repressive effect of atRA persisted, suggesting there are also FXR/RXR-independent mechanisms mediating atRA repression of CYP7A1 expression. Chromatin immunoprecipitation (ChIP) assay and cell transfection results indicate that PGC-1α plays a role in the FXR/RXR-independent mechanism. Our findings may provide a potential explanation for hyperlipidemic side effects observed in some patients treated with high-dose RA. Mammals dispose of cholesterol mainly by converting cholesterol to bile acids where cholesterol 7α-hydroxylase (CYP7A1) is the rate-limiting enzyme (1.Chiang J.Y. Regulation of bile acid synthesis: pathways, nuclear receptors, and mechanisms.J. Hepatol. 2004; 40: 539-551Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar). Genetic deficiency of CYP7A1 in humans leads to hyperlipidemia, including hypercholesterolemia and hypertriglyceridemia (2.Pullinger C.R. Eng C. Salen G. Shefer S. Batta A.K. Erickson S.K. Verhagen A. Rivera C.R. Mulvihill S.J. Malloy M.J. et al.Human cholesterol 7alpha-hydroxylase (CYP7A1) deficiency has a hypercholesterolemic phenotype.J. Clin. Invest. 2002; 110: 109-117Crossref PubMed Scopus (458) Google Scholar). 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However, numerous reports have documented that hyperlipidemia is often a major side effect, including hypercholesterolemia and hypertriglyceridemia (34.Ellis C.N. Swanson N.A. Grekin R.C. Goldstein N.G. Bassett D.R. Anderson T.F. Voorhees J.J. Etretinate therapy causes increases in lipid levels in patients with psoriasis.Arch. Dermatol. 1982; 118: 559-562Crossref PubMed Scopus (44) Google Scholar, 35.Bershad S. Rubinstein A. Paterniti J.R. Le N.A. Poliak S.C. Heller B. Ginsberg H.N. Fleischmajer R. Brown W.V. Changes in plasma lipids and lipoproteins during isotretinoin therapy for acne.N. Engl. J. Med. 1985; 313: 981-985Crossref PubMed Scopus (177) Google Scholar, 36.O'Leary T.J. Simo I.E. Kanigsberg N. Walker J. Goodall J.C. Ooi T.C. Changes in serum lipoproteins and high-density lipoprotein composition during isotretinoin therapy.Clin. Invest. Med. 1987; 10: 355-360PubMed Google Scholar, 37.Fex G.A. Aronsson A. Andersson A. Larsson K. Nilsson-Ehle P. 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Cell culture media (DMEM), fetal bovine serum (FBS), penicillin/streptomycin, trypsin, and PBS were from Invitrogen (Carlsbad, CA). HMM medium is from Lonza (Walkersville, MD). Matrigel were purchased from BD Sciences (Bedford, MA). Luciferase assay kit was purchased from Promega (Madison, WI). [35S]L-Methionine and ECL reagents were purchased from Amersham (Piscataway, NJ). DNA oligos and sequencing were provided by the Keck Biotechnology Center at Yale University. UVI3001 and UVI3003 are gifts from Prof. Angel R. de Lera (Universidade de Vigo, Spain) (41.Nahoum V. Perez E. Germain P. Rodriguez-Barrios F. Manzo F. Kammerer S. Lemaire G. Hirsch O. Royer C.A. Gronemeyer H. et al.Modulators of the structural dynamics of the retinoid X receptor to reveal receptor function.Proc. Natl. Acad. Sci. USA. 2007; 104: 17323-17328Crossref PubMed Scopus (130) Google Scholar). Human FXR (hFXR), RXRα (hRXRα) and I-BABP promoter reporter constructs are described in a previous article (33.Cai S.Y. Xiong L. Wray C.G. Ballatori N. Boyer J.L. The farnesoid X receptor FXRalpha/NR1H4 acquired ligand specificity for bile salts late in vertebrate evolution.Am. J. Physiol. Regul. Integr. Comp. Physiol. 2007; 293: R1400-R1409Crossref PubMed Scopus (43) Google Scholar). Mouse HNF4α and PGC-1α expression plasmids were provided by Dr. Peter Edwards (University of California at Los Angeles, Los Angeles, CA). Human FXR W469A mutant (pCMX-hFXR-W469A) was a gift from Dr. David Mangelsdorf (University of Texas Southwestern Medical Center, Dallas, TX). Mouse Fxr-LBD and TK-gal4 reporter constructs were provided by Dr. David Moore (Baylor College of Medicine, Houston, TX). Constructs pCMX-hFXR-ΔH12, pCMX-hRXRα-ΔC19 were generated by mutating P463X in hFXR and D444X in hRXRα, respectively, using the QuickChange kit (Stratagene, La Jolla, CA). pPac0 and pPacRnLuc plasmids for gene expression in Drosophila S2 cells were provided by Dr. Robert Diasio (University of Alabama, Birmingham, AL). pPac-hFXR and pPac-hRXRα were made by inserting hFXR and hRXRα full-length coding regions into pPac0 vector. GST-FXR LBD and GST-FXR-LBDΔH1-5 expression constructs was made by inserting hFXR LBD or the C-terminal of hFXR LBD (AA341-472) into BamHI and XhoI sites of pGEX-5X-3 vector (Amersham). All mutants were confirmed by DNA sequencing. For the GST-hSRC-1 construct, a 680bp DNA fragment encoding the human steroid receptor coactivator 1 (hSRC-1) amino acid 561-782 was amplified from HepG2 cell cDNA library using PfuUltra DNA polymerase (Stratagene), confirmed by sequencing, and cloned into BamHI and XhoI sites of a pGEX-5X-3 vector. The primers are listed in Table 1.TABLE 1DNA sequence of PCR primers and siRNA oligosNameSequence 5′→ 3′hRXRα-siRNA1TGACGGAGCTTGTGTCCAATThRXRα-siRNA2CAACAAGGACTGCCTGATTTThRXRα-siRNA3GCAAGGACCTGACCTACACTThRXRα-siRNA4GCAAGGACCGGAACGAGAATThFXR P463XCAAGTTTACCTGACTTCTCTGTGAAATCTGGhRXRα D444XGCTCATCGGGTAGACACCCATTGACACCTTCCThFXR-F21GCTGGATCCCGTCTGGGCATTCTGAChFXR-F35GCTGGATCCCAACAAAGTCATGCAGGGhFXR-R22CGCACTCGAGTCACTGCACGTCCCAGATThSRC-1-FGCTGGATCCTCAGGCAGATGAGCTCAChSRC-1-RCGCACTCGAGATCCATCTGTTCTTTCTTTTCC Open table in a new tab Four sets of double-strand, small interference RNA (siRNA) oligos that target human RXRα were chemically synthesized and annealed to duplex by Dharmacon (Chicago, IL) (Table 1). FXR siRNA and control RNA oligo, siCONTROL RISC-Free siRNA (sequence proprietary) were also purchased from Dharmacon. Human hepatocytes were obtained from the Liver Tissue Procurement and Distribution System of the National Institutes of Health (Dr. Stephen Strom, University of Pittsburgh), and maintained on collagen-coated plates using HMM medium (containing dexamethasone and insulin, each 0.1 μM) and overlaid with Matrigel. Purified monoclonal anti-human FGF-19 antibody was purchased from R&D Systems (Minneapolis, MN). HepG2 and HEK293T cells were grown at 37°C in DMEM medium with 10% FBS in 5% CO2 atmosphere. Drosophila S2 cells (kindly provided by Dr. Tian Xu's lab at Yale University) were maintained in Schneider's Drosophila Medium (Invitrogen) supplemented with 10% FBS at 25°C incubation. Lipofectamine 2000 (Invitrogen) was used for transfection of human primary hepatocytes, HepG2, and HEK293T cells, whereas Effectene from Qiagen (Valencia, CA) was used for transfection of Drosophila S2 cells. All cells were transfected in serum-free medium for 16 h. After transfection, cells were treated with specified chemicals for an additional 24 h in serum-free or charcoal-stripped medium. Total RNA was extracted, purified, and converted to cDNA as template. Real-time PCR was performed in an ABI7500 Sequence Detection System. TaqMan primers/probe of GAPDH, SHP, CYP7A1, BSEP, OSTα, and OSTβ are as previously described (42.Cai S.Y. Gautam S. Nguyen T. Soroka C.J. Rahner C. Boyer J.L. ATP8B1 deficiency disrupts the bile canalicular membrane bilayer structure in hepatocytes, but FXR expression and activity are maintained.Gastroenterology. 2009; 136: 1060-1069Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar), whereas primers/probe of FGF19, CYP27A1, and NTCP were purchased from ABI. GAPDH was used as reference for normalizing the data. Protein was extracted from samples and analyzed as previously described (42.Cai S.Y. Gautam S. Nguyen T. Soroka C.J. Rahner C. Boyer J.L. ATP8B1 deficiency disrupts the bile canalicular membrane bilayer structure in hepatocytes, but FXR expression and activity are maintained.Gastroenterology. 2009; 136: 1060-1069Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Polyclonal antibodies against FXR, RXRα, HNF4α, PGC-1α, and SH-PTP were from Santa Cruz and used at dilutions of 1:200, 1:500, 1:150, 1:150, and 1:1000, respectively. For Western blot analysis, total cell lysate (∼50 µg protein) were separated on a 10% SDS-polyacrylamide gel. After electrophoresis, proteins were transferred onto polyvinylidene fluoride (PVDF) microporous membranes (BioRad), the membranes were blocked with 5% nonfat milk, followed by primary antibody and HRP-conjugated secondary antibody incubation. The immune complexes were detected with the enhanced chemiluminescence reagent kit, and the signals were recorded on X-ray film. A dual-luciferase assay was applied for functional characterization by cotransfecting hFXR construct with the pCMX-hRXRα, pGL3-hI-BABP, and phRL-CMV plasmids (constitutively expressing Renilla luciferase) into HEK293T or HepG2 cells as previously described (33.Cai S.Y. Xiong L. Wray C.G. Ballatori N. Boyer J.L. The farnesoid X receptor FXRalpha/NR1H4 acquired ligand specificity for bile salts late in vertebrate evolution.Am. J. Physiol. Regul. Integr. Comp. Physiol. 2007; 293: R1400-R1409Crossref PubMed Scopus (43) Google Scholar). The firefly luciferase activity was normalized by Renilla luciferase activity. Data are presented as mean ± SD of three or more independent experiments, and each transfection was performed in triplicate. The GST, GST-hFXR LBD, GST-hFXR LBDΔH1-5, and GST-hSRC-1 (AA561-782) fusion proteins were expressed in E. coli DH5α and purified using glutathione-Sepharose 4B beads. Different amounts of 3H-atRA were directly incubated with proteins bound on Sepharose 4B beads and analyzed as described previously (43.Yang N. Schule R. Mangelsdorf D.J. Evans R.M. Characterization of DNA binding and retinoic acid binding properties of retinoic acid receptor.Proc. Natl. Acad. Sci. USA. 1991; 88: 3559-3563Crossref PubMed Scopus (111) Google Scholar). 35S-labeled hFXR and hRARα were synthesized using the TNT T7 QuickCoupled transcription/translation kit (Prom
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