Accepted Manuscript Vitamin D Receptor Activation Down-regulates Small Heterodimer Partner and Increases CYP7A1 to Lower Cholesterol Edwin C.Y. Chow, Lilia Magomedova, Holly P. Quach, Rucha H. Patel, Matthew R. Durk, Jianghong Fan, Han-Joo Maeng, Kamdi Irondi, Sayeepriyadarshini Anakk, David D. Moore, Carolyn L. Cummins, K. Sandy Pang PII: DOI: Reference:
S0016-5085(13)01840-4 10.1053/j.gastro.2013.12.027 YGAST 58869
To appear in: Gastroenterology Accepted Date: 17 December 2013 Please cite this article as: Chow ECY, Magomedova L, Quach HP, Patel RH, Durk MR, Fan J, Maeng HJ, Irondi K, Anakk S, Moore DD, Cummins CL, Pang KS, Vitamin D Receptor Activation Down-regulates Small Heterodimer Partner and Increases CYP7A1 to Lower Cholesterol, Gastroenterology (2014), doi: 10.1053/j.gastro.2013.12.027. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. All studies published in Gastroenterology are embargoed until 3PM ET of the day they are published as corrected proofs on-line. Studies cannot be publicized as accepted manuscripts or uncorrected proofs.
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Title: Vitamin D Receptor Activation Down-regulates Small Heterodimer Partner and Increases CYP7A1 to Lower Cholesterol
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Short Title: VDR up-regulates Cyp7a1 and lowers cholesterol Authors: Edwin C.Y. Chow1, Lilia Magomedova1, Holly P. Quach1,†, Rucha H. Patel1,†, Matthew R. Durk1, Jianghong Fan1, Han-Joo Maeng1,3, Kamdi Irondi1, Sayeepriyadarshini Anakk 2,√ , David D. Moore2, Carolyn L. Cummins1,‡, and K. Sandy Pang1,‡ (†,‡contributed equally) 1
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Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, Canada; 2Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas, USA; 3Current Address: College of Pharmacy, Inje University, 607 Obang-dong, Gimhae, Gyeongnam 621-749, South Korea √
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Current address: Department of Molecular & Integrative Physiology, University of UrbanaChampaign, Illinois 61801
Grant support: We gratefully acknowledge support from the Canadian Institutes of Health Research, the Natural Sciences and Engineering Research Council of Canada and the Ontario Graduate Scholarship Program
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Acknowledgement: We thank Drs. Nan Wu and Martin Wagner, Baylor College of Medicine, Texas Medical Center, Houston, for assistance with studies on the bile acid pool sizes of Shp-/mice
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Abbreviations: ApoE, Apolipoprotein E; Asbt, rodent apical sodium dependent bile acid transporter, Bsep, rodent bile salt export pump; Cyp7a1/CYP7A1, rodent/human cholesterol 7αhydroxylase; Fgf15/FGF19, rodent/human fibroblast growth factor 15/19; FGFR, fibroblast growth factor receptor; Fxr/FXR, rodent/human farnesoid X receptor; Gapdh, rodent glyceraldehyde-3-phosphate dehydrogenase; HDL-C, high-density lipoprotein cholesterol; HmgCo-A/HMGCo-A reductase, rodent/human 3-hydroxy-3-methyl-glutaryl-CoA reductase; HNF-4α, hepatocyte nuclear factor 4α; Ibabp, mouse ileal bile acid binding protein; LCA, lithocholic acid; LDL-C, low-density lipoprotein cholesterol; Ldlr, low-density lipoprotein receptor; LRH-1, liver receptor homolog-1; LXRα, liver X receptor alpha; Npc1l1, rodent Niemann-Pick C1-Like 1 transporter; Ntcp, mouse sodium-dependent taurocholate cotransporting polypeptide; Rxr/RXR, rodent/human retinoid X receptor; Shp/SHP, rodent/human small heterodimer partner; Sr-b1, mouse scavenger receptor class B member 1; Vldlr, mouse very low-density lipoprotein receptor; EMSA, electromobility shift assays; ChIP, chromatin immunoprecipitation; DAB, 3,3'-diaminobenzidine tetrahydrochloride; PBS, phosphate buffered saline; HRP, horseradish peroxidase; RLU, relative luciferase units; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid; BSA, bovine serum albumin; HEK293, human embryonic kidney 293
ACCEPTED MANUSCRIPT 2 Correspondence: Dr. K. Sandy Pang, Leslie Dan Faculty of Pharmacy, University of Toronto, 144 College Street, Toronto, Ontario, Canada M5S 3M2. TEL: 416-978-6164, FAX: 416-9788511, E-mail: [email protected] Disclosures: The authors disclose no conflicts.
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Author contributions Edwin Chow: performed most of the in vivo studies and gene profiling, Western blotting; cholesterol and calcium measurements; made the seminal observation that cholesterol lowering was due to SHP inhibition; assayed 1,25(OH)2D3 with EIA; examined temporal changes of 1,25(OH)2D3 vs. changes of genes in liver; wrote first draft of paper and contributed to revisions
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Lilia Magomedova: performed molecular studies on SHP promoter activity, truncation and deletion studies of the hSHP promoter to identify putative VDREs, EMSA, and LCMS assay of bile acids
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Holly P. Quach: conducted studies on Fxr-/- mice fed the normal and Western diets, and treatment with 1,25(OH)2D3; performed gene profiling and Western blotting; teamed up with Edwin in the determination of bile acids and cholesterol, extraction of bile acids for pool size determination and fecal excretion; and EIA for 1,25(OH)2D3 Rucha Patel: conducted mouse primary hepatocyte studies and ChIP assays
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Matthew R. Durk: was responsible for tissue collection and liver immunostaining to identify VDR protein in murine livers; tested specificity of antibody with Vdr-/- mice Jianghong Fan: conducted human hepatocyte experiments Han-Joo Maeng: performed microsomal assay of murine Cyp7a1 activity with HPLC
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Kamdi Irondi: performed the first set of studies on Fxr+/+ and Fxr-/- mice and found that 1,25(OH)2D3 increased Cyp7a1 but not Asbt; observed cholesterol lowering in mice
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Sayeepriyadarshini Anakk: prepared and treated Shp-/- mice, normal diet-fed or Western dietfed and 1,25(OH)2D3 treatment David D. Moore: conceived studies for normal diet-fed and Western diet-fed Shp-/- mice studies and contributed to the writing and revision of paper Carolyn L. Cummins: conceived the molecular studies (promoter activity, EMSA, ChIP) and planned the studies of mice fed the Western diet; contributed significantly to writing, rebuttal and revision of paper K. Sandy Pang: senior author and ultimately responsible for the entire paper; study planning, identification of SHP as key nuclear receptor that suppressed VDR; data interpretation; and writing and revision of the manuscript
ACCEPTED MANUSCRIPT 3 ABSTRACT BACKGROUND & AIMS: Little is known about effects of the vitamin D receptor (VDR) on hepatic activity of CYP7A1 and cholesterol metabolism. We studied these processes in mice and
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mouse and human hepatocytes. METHODS: Fxr-/-, Shp-/-, and C57BL/6 (control) mice were fed normal or Western diets for 3 weeks and then given intraperitoneal injections of 1α,25dihydroxyvitamin D3 [4 doses of 1,25(OH)2D3, 2.5 µg/kg, every other day]. Plasma and tissue
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samples were collected and levels of Vdr, Shp, Cyp7a1, Cyp24a1, and Fgf15 expression, as well as levels of cholesterol, were measured. We studied the regulation of Shp by Vdr using reporter
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and mobility shift assays with transfected HEK293 cells, quantitative PCR with mouse tissues and mouse and human hepatocytes, and chromatin immunoprecipitation (ChIP) assays with mouse liver. RESULTS: We detected Vdr in livers of mice. In mice fed normal diets and given injections of 1,25(OH)2D3, liver and plasma concentrations of 1,25(OH)2D3 increased and
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decreased together. Changes in hepatic Cyp7a1 mRNA correlated with those of Cyp24a1 (a Vdr target gene) and inversely to Shp mRNA, but not ileal Fgf15 mRNA. Similarly, incubation of with 1,25(OH)2D3 increased levels of CYP24A1 and CYP7A1 mRNA in mouse and human
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hepatocytes, and reduced levels of Shp mRNA in mouse hepatocytes. In Fxr-/- and wild-type mice with hypercholesterolemia, injection of 1,25(OH)2D3 consistently reduced levels of plasma
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and liver cholesterol and Shp mRNA, and increased hepatic Cyp7a1 mRNA and protein; these changes were not observed in Shp-/- mice given 1,25(OH)2D3 and fed Western diets. Truncation of the SHP promoter and deletion analyses revealed VDR-dependent inhibition of SHP, whereas mobility shift assays showed direct binding of VDR to enhancer regions of SHP. Moreover, ChIP analysis of livers from mice showed that injection of 1,25(OH)2D3 increased recruitment of Vdr
ACCEPTED MANUSCRIPT 4 and Rxr to the Shp promoter. Conclusion: In mouse and human hepatocytes, activation of the VDR represses hepatic SHP to increase levels of CYP7A1 and reduce cholesterol.
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KEYWORDS: liver, bile acid, farnesoid X receptor, transcriptional regulation
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Cholesterol is an essential component of cell membranes and the precursor to steroid hormones and bile acids. In excess, cholesterol can lead to atherosclerosis and coronary heart disease. In
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liver, cholesterol is metabolized to bile acids by cholesterol 7α-hydroxylase (CYP7A1), which is the rate-limiting metabolic enzyme in the classic bile acid synthetic pathway.1 The CYP7A1 promoter contains highly conserved bile acid responsive regions known to be modulated by the feedback repression by various transcription factors in response to increasing hepatic bile acid
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concentrations.1 A primary, negative feedback mechanism of CYP7A1 regulation is the farnesoid
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X receptor (FXR, NR1H4) and small heterodimer partner (SHP, NR0B2) regulatory cascade.2 Bile acids such as chenodeoxycholic acid (CDCA) activate FXR to increase transcription of SHP, an atypical nuclear receptor that lacks a DNA binding domain and represses CYP7A1 activation by suppression of transcription factors, the liver receptor homolog-1 (LRH-1, NR5A2) and hepatocyte nuclear factor 4α (HNF-4α, NR2A1), which are essential for CYP7A1 expression.3 A
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second negative feedback mechanism on CYP7A1 is found in the intestine, where activation of FXR induces the fibroblast growth factor 15/19 (rodent/human), a hormonal signaling molecule that represses CYP7A1 through interaction with the liver fibroblast growth factor receptor 4
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(FGFR4) via the c-Jun signaling pathway.4
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The vitamin D receptor (VDR, NR1I1) binds to its endogenous ligand, 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3] or lithocholic acid (LCA, alternate VDR ligand)5 to activate the transcription of genes. Various mechanisms implicate a role for VDR on CYP7A1 regulation.6 Activation of VDR was found to antagonize the CDCA-dependent transactivation of FXR in VDR-transfected HepG2 cells7 and blunt the Lxrα-mediated induction of Cyp7a1 mRNA in rat hepatoma cells.8 VDR inhibition of CYP7A1 transcription in human hepatocytes and HepG2 cells has been attributed to blockage of HNF-4α-mediated activation of CYP7A1.9 Induction of intestinal Fgf15
ACCEPTED MANUSCRIPT 6 following a high dose of 1,25(OH)2D3 in mice was shown to down-regulate Cyp7a1 mRNA level.10 By contrast, Vdr-knockout mice are reported to have higher total serum cholesterol,11 and treatment with 1α-hydroxyvitamin D3, a potent 1,25(OH)2D3 precursor, up-regulated Cyp7a1
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mRNA expression in mice.12,13 Likewise, doxercalciferol, a vitamin D analogue, decreased the accumulation of triglycerides and cholesterol in murine kidney.14 In rat, 1,25(OH)2D3 downregulated liver Cyp7a1 by an indirect mechanism.15 The divergent views are partially reconciled
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by species differences, with VDR protein levels being extremely low in rat liver, but present at detectable levels in mouse and man.16 The down-regulation of Cyp7a1 after 1,25(OH)2D3
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treatment in the rat is explained as a secondary hepatic Fxr and not Vdr effect, since increased bile acid absorption into portal blood occurred as a result of Vdr-mediated induction of the intestinal apical sodium-dependent bile acid transporter (Asbt).15 Clinical reports relating vitamin D status to cholesterol [cholesterol levels vs. 1,25(OH)2D3 or its less active precursor, 25-
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hydroxyvitamin D3, 25(OH)D3] are equivocal. Both short17 and long18 term vitamin D treatment did not improve lipid profiles nor lower cholesterol and only resulted in slightly lower serum triglyceride, whereas other studies documented increased HDL-C,19 or decreased total cholesterol
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and triglyceride but not changed HDL-C and LDL-C levels.20 A recent, population-based study established an association between 1,25(OH)2D3 and HDL-C and 25(OH)D3 and total cholesterol,
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LDL-C, and triglyceride.21 Atorvastatin, supplemented with vitamin D, further lowered total cholesterol and LDL-C,22 and patients receiving statin or niacin supplemented with vitamin D and fish oil showed reduced LDL-C and triglycerides and elevated HDL-C.23 Hence, the role of the VDR in liver cholesterol regulation remains controversial. The intent of this study was to clarify the impact of Vdr on Cyp7a1 regulation and cholesterol lowering. We verified the presence of Vdr in murine liver, and demonstrated that 1,25(OH)2D3
ACCEPTED MANUSCRIPT 7 given to mice rapidly reached the liver, resulting in induction of hepatic Cyp7a1 via downregulation of Shp. We showed that Vdr-mediated Shp repression and up-regulation of Cyp7a1 was Fxr-independent but Shp-dependent in vivo. Luciferase reporter, EMSA, and ChIP assays
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further supported a direct role for Vdr/VDR in the repression of Shp/SHP to result in upregulation of Cyp7a1/CYP7A1, thus providing a novel mechanism for cholesterol lowering. In mouse and human hepatocytes, Cyp7a1/CYP7A1 expression was elevated upon exposure to
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1,25(OH)2D3.
ACCEPTED MANUSCRIPT 8 METHODS In Supplemental Methods, we provided information on materials, mouse strains, antibodies,
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plasmids and procedures for real-time PCR, Western blotting, microsomal preparation for Cyp7a1 activity, and assay procedures for bile acid pool size, cholesterol, and 1,25(OH)2D3. Immunostaining of murine liver Vdr. Livers of male C57BL/6 mice were perfusion-fixed with
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PBS and 4% paraformaldehyde, and kept overnight at 4°C. Then, 7 µm thick paraffin-embedded sections were dewaxed and incubated in 2N HCl at 37°C for 30 min. Sections were pre-blocked
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with 5% goat serum in PBS containing 0.1% Tween-20, then incubated with the primary antiVDR antibody 9A7 (1:50 v/v) overnight. After rinsing 3x with 5% goat serum in PBS containing 0.1% Tween-20, the secondary goat anti-rat HRP antibody was added for 2 h at room temperature and visualized using a metal-enhanced DAB substrate kit (Thermo Scientific, Rockford, IL).
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After several washes, sections were imaged using a Nikon E1000R microscope. 1,25(OH)2D3 treatment of mice in vivo. In each set of in vivo studies, doses of 0 or 2.5 µg/kg 1,25(OH)2D3, in sterile corn oil, were given intraperitoneally (ip) every other day for 8 days (q2d
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x4; Day 0 to Day 8) at 9-10 a.m.24 First, we treated normal diet-fed, male C57BL/6 [wildtype or Fxr+/+] and Fxr-/- mice (8 to 12 weeks; n=4-10) with 1,25(OH)2D3, and blood and tissues were
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harvested on Day 8 between 12-2 p.m. In the second study, blood samples and livers from wildtype mice were collected at different time points during the 1,25(OH)2D3 treatment period, as described.25 In the third study, hypercholesterolemic models comprising of wildtype, Fxr-/- and Shp-/- mice (6-8 weeks old; n = 4-10) fed a Western diet [Harlan Teklad Cat#88137; high fat (42%)/high cholesterol (0.2%) diet] for 3 weeks were established, a period during which Cyp7a1 mRNA/protein expression was unchanged. These mice were given four repeated doses of 1,25(OH)2D3 (q2d x4; Day 0 to Day 8) at the beginning of the 3rd week of the Western diet. On
ACCEPTED MANUSCRIPT 9 Day 8, systemic and portal blood samples and tissues were obtained under anesthesia.15,24 Basal mRNA levels of intestinal and liver genes in Fxr-/- and Shp-/- mice were compared to those of wildtype mice (Supplemental Figures 1A and 1B). 1,25(OH)2D3 treatment resulted in a slight
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elevation of plasma calcium but relatively unchanged phosphorous, ALT and portal bile acid levels (Supplemental Table 1).
Mouse primary hepatocytes. Mouse primary hepatocytes were isolated26 and treated with vehicle
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(0.1% EtOH) or 100 nM 1,25(OH)2D3 (M199 media without FBS) for 0, 3, 6, 9, 12, and 24 h. Cells were harvested for mRNA determination at 9 h, the minimal time required for induction of
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Cyp24a1.27 Dose-dependent activation of Cyp24a1 by 1,25(OH)2D3 (10 to 250 nM) was observed (data not shown).
Human hepatocytes. Upon arrival, the cold preservation medium for storage of human primary hepatocytes (gift from Dr. Jas Sahi, Life Technologies, NY) was replaced with Williams E
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medium, supplemented with the cell maintenance cocktail [0.1 mM of dexamethasone, 50 U/ml penicillin and 50 µg/ml of streptomycin, 2 mM of L-glutamine, 10 µM of HEPES, and ITS+ (6.25 g/ml insulin, 6.25 g/ml transferrin, 6.25 g/ml selenous acid, 1.25 mg/ml BSA and 5.33 g/ml
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linoleic acid)]. Plates (6-well; 2.1 x 106 cells/cm2) were acclimatized overnight at 37 °C in a
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humidified incubator. On the next day, cells were treated vehicle (0.1% ethanol) or 100 nM 1,25(OH)2D3 and harvested at 3, 6, 12 and 24 h. Activation of VDR was confirmed by CYP24A1 mRNA induction.
Transfection assays of HEK293 cells. Cell transfection was performed in media containing 10%charcoal-stripped FBS using calcium phosphate in 96-well plates. The total amount of plasmid DNA (150 ng/well) included 50 ng reporter, 20 ng pCMX-β-galactosidase, 15 ng nuclear receptor, 15 ng pCMX-Lrh-1 and pGEM filler plasmid. Ligands were added at 6–8 h post-
ACCEPTED MANUSCRIPT 10 transfection. Cells harvested 14–16 h later were assayed for luciferase and β-galactosidase activity. Luciferase values were normalized to β-galactosidase to control for transfection
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efficiency and expressed as Relative Luciferase Units (RLU). Nuclear protein extracts. HEK293 cells were transfected with 10 µg Vdr, Rxrα or CMX plasmid DNA using calcium phosphate in 10 mm plates. At 30 h post-transfection, nuclear protein
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extracts were prepared.26
Electrophoretic mobility shift assay (EMSA). Oligonucleotide sequences are described in
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Supplemental Table 3. Oligos were biotinylated using a biotin 3′ end DNA labeling kit (Pierce, Rockford, IL). DNA binding reactions consisted of 2.5 µl of NE-PER nuclear extracts in binding buffer [10 mM Tris, 50 mM KCl, 1 mM DTT, 1 mM EDTA, 5 % glycerol, 1 µg BSA and 1 µg poly(dI-dC); pH 7.5]. After a 20 min pre-incubation, 50 fmol of annealed biotinylated DNA was
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added and incubated for 30 min on ice. Reactions were loaded onto a 6% polyacrylamide gel, transferred to Biodyne B membrane (Pierce) and crosslinked. Detection was carried out using the Light Shift Chemiluminescent EMSA Kit (Pierce). ChIP assays were performed on frozen livers following vehicle- or 1,25(OH)2D3-
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ChIP.
treatement in normal diet-fed wildtype mice (3-4 sets of experiment, in triplicates) at 12 h after
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the 4th injection, as described.26 Liver nuclei were re-suspended in 2x pellet volume of sonication buffer (0.2% SDS, 1% Triton-X, 0.1% Na-deoxycholate, 1 mM EDTA and 50 mM Tris-HCl, pH 8.0, 150 mM NaCl and protease inhibitors). Chromatin, diluted 2-fold in buffer (sonication buffer without SDS) and 400 µl of diluted sample, was incubated with 10 µg Vdr, Rxrα, Lrh-1 or 2 µg of
H3K9me3
antibodies
(see
Supplemental
Methods)
overnight
and
used
for
immunoprecipitation. Following spin column purification, the eluate was diluted 5-fold with water, and qPCR was performed using 5 µl of template DNA with the following primer set:
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5’-GAGCGCCTGAGACCTTGGT-3’
and
Reverse:
5’-
TCAAGTGCATAAACAGGGTCATTAA-3’ amplifying the putative mouse Shp VDRE. Quantification was performed by qPCR (standard curve method) using serial dilutions of the
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input as standards.
Statistics. Data are expressed as mean ± S.E.M. for in vivo data and mean ± S.D. for in vitro data. For comparison of in vivo and in vitro data between two groups, the Mann-Whitney U and the
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unpaired Student’s t tests were used, respectively, and P
S0016-5085(13)01840-4 10.1053/j.gastro.2013.12.027 YGAST 58869
To appear in: Gastroenterology Accepted Date: 17 December 2013 Please cite this article as: Chow ECY, Magomedova L, Quach HP, Patel RH, Durk MR, Fan J, Maeng HJ, Irondi K, Anakk S, Moore DD, Cummins CL, Pang KS, Vitamin D Receptor Activation Down-regulates Small Heterodimer Partner and Increases CYP7A1 to Lower Cholesterol, Gastroenterology (2014), doi: 10.1053/j.gastro.2013.12.027. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. All studies published in Gastroenterology are embargoed until 3PM ET of the day they are published as corrected proofs on-line. Studies cannot be publicized as accepted manuscripts or uncorrected proofs.
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Title: Vitamin D Receptor Activation Down-regulates Small Heterodimer Partner and Increases CYP7A1 to Lower Cholesterol
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Short Title: VDR up-regulates Cyp7a1 and lowers cholesterol Authors: Edwin C.Y. Chow1, Lilia Magomedova1, Holly P. Quach1,†, Rucha H. Patel1,†, Matthew R. Durk1, Jianghong Fan1, Han-Joo Maeng1,3, Kamdi Irondi1, Sayeepriyadarshini Anakk 2,√ , David D. Moore2, Carolyn L. Cummins1,‡, and K. Sandy Pang1,‡ (†,‡contributed equally) 1
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Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, Canada; 2Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas, USA; 3Current Address: College of Pharmacy, Inje University, 607 Obang-dong, Gimhae, Gyeongnam 621-749, South Korea √
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Current address: Department of Molecular & Integrative Physiology, University of UrbanaChampaign, Illinois 61801
Grant support: We gratefully acknowledge support from the Canadian Institutes of Health Research, the Natural Sciences and Engineering Research Council of Canada and the Ontario Graduate Scholarship Program
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Acknowledgement: We thank Drs. Nan Wu and Martin Wagner, Baylor College of Medicine, Texas Medical Center, Houston, for assistance with studies on the bile acid pool sizes of Shp-/mice
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Abbreviations: ApoE, Apolipoprotein E; Asbt, rodent apical sodium dependent bile acid transporter, Bsep, rodent bile salt export pump; Cyp7a1/CYP7A1, rodent/human cholesterol 7αhydroxylase; Fgf15/FGF19, rodent/human fibroblast growth factor 15/19; FGFR, fibroblast growth factor receptor; Fxr/FXR, rodent/human farnesoid X receptor; Gapdh, rodent glyceraldehyde-3-phosphate dehydrogenase; HDL-C, high-density lipoprotein cholesterol; HmgCo-A/HMGCo-A reductase, rodent/human 3-hydroxy-3-methyl-glutaryl-CoA reductase; HNF-4α, hepatocyte nuclear factor 4α; Ibabp, mouse ileal bile acid binding protein; LCA, lithocholic acid; LDL-C, low-density lipoprotein cholesterol; Ldlr, low-density lipoprotein receptor; LRH-1, liver receptor homolog-1; LXRα, liver X receptor alpha; Npc1l1, rodent Niemann-Pick C1-Like 1 transporter; Ntcp, mouse sodium-dependent taurocholate cotransporting polypeptide; Rxr/RXR, rodent/human retinoid X receptor; Shp/SHP, rodent/human small heterodimer partner; Sr-b1, mouse scavenger receptor class B member 1; Vldlr, mouse very low-density lipoprotein receptor; EMSA, electromobility shift assays; ChIP, chromatin immunoprecipitation; DAB, 3,3'-diaminobenzidine tetrahydrochloride; PBS, phosphate buffered saline; HRP, horseradish peroxidase; RLU, relative luciferase units; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid; BSA, bovine serum albumin; HEK293, human embryonic kidney 293
ACCEPTED MANUSCRIPT 2 Correspondence: Dr. K. Sandy Pang, Leslie Dan Faculty of Pharmacy, University of Toronto, 144 College Street, Toronto, Ontario, Canada M5S 3M2. TEL: 416-978-6164, FAX: 416-9788511, E-mail: [email protected] Disclosures: The authors disclose no conflicts.
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Author contributions Edwin Chow: performed most of the in vivo studies and gene profiling, Western blotting; cholesterol and calcium measurements; made the seminal observation that cholesterol lowering was due to SHP inhibition; assayed 1,25(OH)2D3 with EIA; examined temporal changes of 1,25(OH)2D3 vs. changes of genes in liver; wrote first draft of paper and contributed to revisions
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Lilia Magomedova: performed molecular studies on SHP promoter activity, truncation and deletion studies of the hSHP promoter to identify putative VDREs, EMSA, and LCMS assay of bile acids
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Holly P. Quach: conducted studies on Fxr-/- mice fed the normal and Western diets, and treatment with 1,25(OH)2D3; performed gene profiling and Western blotting; teamed up with Edwin in the determination of bile acids and cholesterol, extraction of bile acids for pool size determination and fecal excretion; and EIA for 1,25(OH)2D3 Rucha Patel: conducted mouse primary hepatocyte studies and ChIP assays
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Matthew R. Durk: was responsible for tissue collection and liver immunostaining to identify VDR protein in murine livers; tested specificity of antibody with Vdr-/- mice Jianghong Fan: conducted human hepatocyte experiments Han-Joo Maeng: performed microsomal assay of murine Cyp7a1 activity with HPLC
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Kamdi Irondi: performed the first set of studies on Fxr+/+ and Fxr-/- mice and found that 1,25(OH)2D3 increased Cyp7a1 but not Asbt; observed cholesterol lowering in mice
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Sayeepriyadarshini Anakk: prepared and treated Shp-/- mice, normal diet-fed or Western dietfed and 1,25(OH)2D3 treatment David D. Moore: conceived studies for normal diet-fed and Western diet-fed Shp-/- mice studies and contributed to the writing and revision of paper Carolyn L. Cummins: conceived the molecular studies (promoter activity, EMSA, ChIP) and planned the studies of mice fed the Western diet; contributed significantly to writing, rebuttal and revision of paper K. Sandy Pang: senior author and ultimately responsible for the entire paper; study planning, identification of SHP as key nuclear receptor that suppressed VDR; data interpretation; and writing and revision of the manuscript
ACCEPTED MANUSCRIPT 3 ABSTRACT BACKGROUND & AIMS: Little is known about effects of the vitamin D receptor (VDR) on hepatic activity of CYP7A1 and cholesterol metabolism. We studied these processes in mice and
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mouse and human hepatocytes. METHODS: Fxr-/-, Shp-/-, and C57BL/6 (control) mice were fed normal or Western diets for 3 weeks and then given intraperitoneal injections of 1α,25dihydroxyvitamin D3 [4 doses of 1,25(OH)2D3, 2.5 µg/kg, every other day]. Plasma and tissue
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samples were collected and levels of Vdr, Shp, Cyp7a1, Cyp24a1, and Fgf15 expression, as well as levels of cholesterol, were measured. We studied the regulation of Shp by Vdr using reporter
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and mobility shift assays with transfected HEK293 cells, quantitative PCR with mouse tissues and mouse and human hepatocytes, and chromatin immunoprecipitation (ChIP) assays with mouse liver. RESULTS: We detected Vdr in livers of mice. In mice fed normal diets and given injections of 1,25(OH)2D3, liver and plasma concentrations of 1,25(OH)2D3 increased and
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decreased together. Changes in hepatic Cyp7a1 mRNA correlated with those of Cyp24a1 (a Vdr target gene) and inversely to Shp mRNA, but not ileal Fgf15 mRNA. Similarly, incubation of with 1,25(OH)2D3 increased levels of CYP24A1 and CYP7A1 mRNA in mouse and human
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hepatocytes, and reduced levels of Shp mRNA in mouse hepatocytes. In Fxr-/- and wild-type mice with hypercholesterolemia, injection of 1,25(OH)2D3 consistently reduced levels of plasma
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and liver cholesterol and Shp mRNA, and increased hepatic Cyp7a1 mRNA and protein; these changes were not observed in Shp-/- mice given 1,25(OH)2D3 and fed Western diets. Truncation of the SHP promoter and deletion analyses revealed VDR-dependent inhibition of SHP, whereas mobility shift assays showed direct binding of VDR to enhancer regions of SHP. Moreover, ChIP analysis of livers from mice showed that injection of 1,25(OH)2D3 increased recruitment of Vdr
ACCEPTED MANUSCRIPT 4 and Rxr to the Shp promoter. Conclusion: In mouse and human hepatocytes, activation of the VDR represses hepatic SHP to increase levels of CYP7A1 and reduce cholesterol.
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KEYWORDS: liver, bile acid, farnesoid X receptor, transcriptional regulation
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Cholesterol is an essential component of cell membranes and the precursor to steroid hormones and bile acids. In excess, cholesterol can lead to atherosclerosis and coronary heart disease. In
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liver, cholesterol is metabolized to bile acids by cholesterol 7α-hydroxylase (CYP7A1), which is the rate-limiting metabolic enzyme in the classic bile acid synthetic pathway.1 The CYP7A1 promoter contains highly conserved bile acid responsive regions known to be modulated by the feedback repression by various transcription factors in response to increasing hepatic bile acid
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concentrations.1 A primary, negative feedback mechanism of CYP7A1 regulation is the farnesoid
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X receptor (FXR, NR1H4) and small heterodimer partner (SHP, NR0B2) regulatory cascade.2 Bile acids such as chenodeoxycholic acid (CDCA) activate FXR to increase transcription of SHP, an atypical nuclear receptor that lacks a DNA binding domain and represses CYP7A1 activation by suppression of transcription factors, the liver receptor homolog-1 (LRH-1, NR5A2) and hepatocyte nuclear factor 4α (HNF-4α, NR2A1), which are essential for CYP7A1 expression.3 A
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second negative feedback mechanism on CYP7A1 is found in the intestine, where activation of FXR induces the fibroblast growth factor 15/19 (rodent/human), a hormonal signaling molecule that represses CYP7A1 through interaction with the liver fibroblast growth factor receptor 4
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(FGFR4) via the c-Jun signaling pathway.4
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The vitamin D receptor (VDR, NR1I1) binds to its endogenous ligand, 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3] or lithocholic acid (LCA, alternate VDR ligand)5 to activate the transcription of genes. Various mechanisms implicate a role for VDR on CYP7A1 regulation.6 Activation of VDR was found to antagonize the CDCA-dependent transactivation of FXR in VDR-transfected HepG2 cells7 and blunt the Lxrα-mediated induction of Cyp7a1 mRNA in rat hepatoma cells.8 VDR inhibition of CYP7A1 transcription in human hepatocytes and HepG2 cells has been attributed to blockage of HNF-4α-mediated activation of CYP7A1.9 Induction of intestinal Fgf15
ACCEPTED MANUSCRIPT 6 following a high dose of 1,25(OH)2D3 in mice was shown to down-regulate Cyp7a1 mRNA level.10 By contrast, Vdr-knockout mice are reported to have higher total serum cholesterol,11 and treatment with 1α-hydroxyvitamin D3, a potent 1,25(OH)2D3 precursor, up-regulated Cyp7a1
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mRNA expression in mice.12,13 Likewise, doxercalciferol, a vitamin D analogue, decreased the accumulation of triglycerides and cholesterol in murine kidney.14 In rat, 1,25(OH)2D3 downregulated liver Cyp7a1 by an indirect mechanism.15 The divergent views are partially reconciled
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by species differences, with VDR protein levels being extremely low in rat liver, but present at detectable levels in mouse and man.16 The down-regulation of Cyp7a1 after 1,25(OH)2D3
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treatment in the rat is explained as a secondary hepatic Fxr and not Vdr effect, since increased bile acid absorption into portal blood occurred as a result of Vdr-mediated induction of the intestinal apical sodium-dependent bile acid transporter (Asbt).15 Clinical reports relating vitamin D status to cholesterol [cholesterol levels vs. 1,25(OH)2D3 or its less active precursor, 25-
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hydroxyvitamin D3, 25(OH)D3] are equivocal. Both short17 and long18 term vitamin D treatment did not improve lipid profiles nor lower cholesterol and only resulted in slightly lower serum triglyceride, whereas other studies documented increased HDL-C,19 or decreased total cholesterol
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and triglyceride but not changed HDL-C and LDL-C levels.20 A recent, population-based study established an association between 1,25(OH)2D3 and HDL-C and 25(OH)D3 and total cholesterol,
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LDL-C, and triglyceride.21 Atorvastatin, supplemented with vitamin D, further lowered total cholesterol and LDL-C,22 and patients receiving statin or niacin supplemented with vitamin D and fish oil showed reduced LDL-C and triglycerides and elevated HDL-C.23 Hence, the role of the VDR in liver cholesterol regulation remains controversial. The intent of this study was to clarify the impact of Vdr on Cyp7a1 regulation and cholesterol lowering. We verified the presence of Vdr in murine liver, and demonstrated that 1,25(OH)2D3
ACCEPTED MANUSCRIPT 7 given to mice rapidly reached the liver, resulting in induction of hepatic Cyp7a1 via downregulation of Shp. We showed that Vdr-mediated Shp repression and up-regulation of Cyp7a1 was Fxr-independent but Shp-dependent in vivo. Luciferase reporter, EMSA, and ChIP assays
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further supported a direct role for Vdr/VDR in the repression of Shp/SHP to result in upregulation of Cyp7a1/CYP7A1, thus providing a novel mechanism for cholesterol lowering. In mouse and human hepatocytes, Cyp7a1/CYP7A1 expression was elevated upon exposure to
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1,25(OH)2D3.
ACCEPTED MANUSCRIPT 8 METHODS In Supplemental Methods, we provided information on materials, mouse strains, antibodies,
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plasmids and procedures for real-time PCR, Western blotting, microsomal preparation for Cyp7a1 activity, and assay procedures for bile acid pool size, cholesterol, and 1,25(OH)2D3. Immunostaining of murine liver Vdr. Livers of male C57BL/6 mice were perfusion-fixed with
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PBS and 4% paraformaldehyde, and kept overnight at 4°C. Then, 7 µm thick paraffin-embedded sections were dewaxed and incubated in 2N HCl at 37°C for 30 min. Sections were pre-blocked
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with 5% goat serum in PBS containing 0.1% Tween-20, then incubated with the primary antiVDR antibody 9A7 (1:50 v/v) overnight. After rinsing 3x with 5% goat serum in PBS containing 0.1% Tween-20, the secondary goat anti-rat HRP antibody was added for 2 h at room temperature and visualized using a metal-enhanced DAB substrate kit (Thermo Scientific, Rockford, IL).
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After several washes, sections were imaged using a Nikon E1000R microscope. 1,25(OH)2D3 treatment of mice in vivo. In each set of in vivo studies, doses of 0 or 2.5 µg/kg 1,25(OH)2D3, in sterile corn oil, were given intraperitoneally (ip) every other day for 8 days (q2d
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x4; Day 0 to Day 8) at 9-10 a.m.24 First, we treated normal diet-fed, male C57BL/6 [wildtype or Fxr+/+] and Fxr-/- mice (8 to 12 weeks; n=4-10) with 1,25(OH)2D3, and blood and tissues were
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harvested on Day 8 between 12-2 p.m. In the second study, blood samples and livers from wildtype mice were collected at different time points during the 1,25(OH)2D3 treatment period, as described.25 In the third study, hypercholesterolemic models comprising of wildtype, Fxr-/- and Shp-/- mice (6-8 weeks old; n = 4-10) fed a Western diet [Harlan Teklad Cat#88137; high fat (42%)/high cholesterol (0.2%) diet] for 3 weeks were established, a period during which Cyp7a1 mRNA/protein expression was unchanged. These mice were given four repeated doses of 1,25(OH)2D3 (q2d x4; Day 0 to Day 8) at the beginning of the 3rd week of the Western diet. On
ACCEPTED MANUSCRIPT 9 Day 8, systemic and portal blood samples and tissues were obtained under anesthesia.15,24 Basal mRNA levels of intestinal and liver genes in Fxr-/- and Shp-/- mice were compared to those of wildtype mice (Supplemental Figures 1A and 1B). 1,25(OH)2D3 treatment resulted in a slight
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elevation of plasma calcium but relatively unchanged phosphorous, ALT and portal bile acid levels (Supplemental Table 1).
Mouse primary hepatocytes. Mouse primary hepatocytes were isolated26 and treated with vehicle
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(0.1% EtOH) or 100 nM 1,25(OH)2D3 (M199 media without FBS) for 0, 3, 6, 9, 12, and 24 h. Cells were harvested for mRNA determination at 9 h, the minimal time required for induction of
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Cyp24a1.27 Dose-dependent activation of Cyp24a1 by 1,25(OH)2D3 (10 to 250 nM) was observed (data not shown).
Human hepatocytes. Upon arrival, the cold preservation medium for storage of human primary hepatocytes (gift from Dr. Jas Sahi, Life Technologies, NY) was replaced with Williams E
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medium, supplemented with the cell maintenance cocktail [0.1 mM of dexamethasone, 50 U/ml penicillin and 50 µg/ml of streptomycin, 2 mM of L-glutamine, 10 µM of HEPES, and ITS+ (6.25 g/ml insulin, 6.25 g/ml transferrin, 6.25 g/ml selenous acid, 1.25 mg/ml BSA and 5.33 g/ml
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linoleic acid)]. Plates (6-well; 2.1 x 106 cells/cm2) were acclimatized overnight at 37 °C in a
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humidified incubator. On the next day, cells were treated vehicle (0.1% ethanol) or 100 nM 1,25(OH)2D3 and harvested at 3, 6, 12 and 24 h. Activation of VDR was confirmed by CYP24A1 mRNA induction.
Transfection assays of HEK293 cells. Cell transfection was performed in media containing 10%charcoal-stripped FBS using calcium phosphate in 96-well plates. The total amount of plasmid DNA (150 ng/well) included 50 ng reporter, 20 ng pCMX-β-galactosidase, 15 ng nuclear receptor, 15 ng pCMX-Lrh-1 and pGEM filler plasmid. Ligands were added at 6–8 h post-
ACCEPTED MANUSCRIPT 10 transfection. Cells harvested 14–16 h later were assayed for luciferase and β-galactosidase activity. Luciferase values were normalized to β-galactosidase to control for transfection
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efficiency and expressed as Relative Luciferase Units (RLU). Nuclear protein extracts. HEK293 cells were transfected with 10 µg Vdr, Rxrα or CMX plasmid DNA using calcium phosphate in 10 mm plates. At 30 h post-transfection, nuclear protein
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extracts were prepared.26
Electrophoretic mobility shift assay (EMSA). Oligonucleotide sequences are described in
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Supplemental Table 3. Oligos were biotinylated using a biotin 3′ end DNA labeling kit (Pierce, Rockford, IL). DNA binding reactions consisted of 2.5 µl of NE-PER nuclear extracts in binding buffer [10 mM Tris, 50 mM KCl, 1 mM DTT, 1 mM EDTA, 5 % glycerol, 1 µg BSA and 1 µg poly(dI-dC); pH 7.5]. After a 20 min pre-incubation, 50 fmol of annealed biotinylated DNA was
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added and incubated for 30 min on ice. Reactions were loaded onto a 6% polyacrylamide gel, transferred to Biodyne B membrane (Pierce) and crosslinked. Detection was carried out using the Light Shift Chemiluminescent EMSA Kit (Pierce). ChIP assays were performed on frozen livers following vehicle- or 1,25(OH)2D3-
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ChIP.
treatement in normal diet-fed wildtype mice (3-4 sets of experiment, in triplicates) at 12 h after
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the 4th injection, as described.26 Liver nuclei were re-suspended in 2x pellet volume of sonication buffer (0.2% SDS, 1% Triton-X, 0.1% Na-deoxycholate, 1 mM EDTA and 50 mM Tris-HCl, pH 8.0, 150 mM NaCl and protease inhibitors). Chromatin, diluted 2-fold in buffer (sonication buffer without SDS) and 400 µl of diluted sample, was incubated with 10 µg Vdr, Rxrα, Lrh-1 or 2 µg of
H3K9me3
antibodies
(see
Supplemental
Methods)
overnight
and
used
for
immunoprecipitation. Following spin column purification, the eluate was diluted 5-fold with water, and qPCR was performed using 5 µl of template DNA with the following primer set:
ACCEPTED MANUSCRIPT 11 Forward:
5’-GAGCGCCTGAGACCTTGGT-3’
and
Reverse:
5’-
TCAAGTGCATAAACAGGGTCATTAA-3’ amplifying the putative mouse Shp VDRE. Quantification was performed by qPCR (standard curve method) using serial dilutions of the
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input as standards.
Statistics. Data are expressed as mean ± S.E.M. for in vivo data and mean ± S.D. for in vitro data. For comparison of in vivo and in vitro data between two groups, the Mann-Whitney U and the
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unpaired Student’s t tests were used, respectively, and P
