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• W2071489706 abstract "The acute phase response is associated with changes in the hepatic expression of genes involved in lipid metabolism. Nuclear hormone receptors that heterodimerize with retinoid X receptor (RXR), such as thyroid receptors, peroxisome proliferator-activated receptors, and liver X receptors, modulate lipid metabolism. We recently demonstrated that these nuclear hormone receptors are repressed during the acute phase response induced by lipopolysaccharide (LPS), consistent with the known decreases in genes that they regulate. In the present study, we show that LPS significantly decreases farnesoid X receptor (FXR) mRNA in mouse liver as early as 8 h after LPS administration, and this decrease was dose-dependent with the half-maximal effect observed at 0.5 μg/100 g of body weight. Gel-shift experiments demonstrated that DNA binding activity to an FXR response element (IR1) is significantly reduced by LPS treatment. Supershift experiments demonstrated that the shifted protein-DNA complex contains FXR and RXR. Furthermore, the expression of FXR target genes, SHP and apoCII, were significantly reduced by LPS (70 and 60%, respectively). Also, LPS decreases hepatic LRH expression in mouse, which may explain the reduced expression of CYP7A1 in the face of SHP repression. In Hep3B human hepatoma cells, both tumor necrosis factor (TNF) and interleukin-1 (IL-1) significantly decreased FXR mRNA, whereas IL-6 did not have any effect. TNF and IL-1 also decreased the DNA binding activity to an IR1 response element and the expression of SHP and apoCII. Importantly, TNF and IL-1 almost completely blocked the expression of luciferase activity linked to a FXR response element promoter construct transfected into Hep3B cells. Together with our earlier studies on the repression of RXRs, peroxisome proliferator-activated receptors, LXRs, thyroid receptors, constitutive androstane receptor, and pregnane X receptor, these results suggest that decreases in nuclear hormone receptors are major contributors to the decreased gene expression that occurs in the negative acute phase response. The acute phase response is associated with changes in the hepatic expression of genes involved in lipid metabolism. Nuclear hormone receptors that heterodimerize with retinoid X receptor (RXR), such as thyroid receptors, peroxisome proliferator-activated receptors, and liver X receptors, modulate lipid metabolism. We recently demonstrated that these nuclear hormone receptors are repressed during the acute phase response induced by lipopolysaccharide (LPS), consistent with the known decreases in genes that they regulate. In the present study, we show that LPS significantly decreases farnesoid X receptor (FXR) mRNA in mouse liver as early as 8 h after LPS administration, and this decrease was dose-dependent with the half-maximal effect observed at 0.5 μg/100 g of body weight. Gel-shift experiments demonstrated that DNA binding activity to an FXR response element (IR1) is significantly reduced by LPS treatment. Supershift experiments demonstrated that the shifted protein-DNA complex contains FXR and RXR. Furthermore, the expression of FXR target genes, SHP and apoCII, were significantly reduced by LPS (70 and 60%, respectively). Also, LPS decreases hepatic LRH expression in mouse, which may explain the reduced expression of CYP7A1 in the face of SHP repression. In Hep3B human hepatoma cells, both tumor necrosis factor (TNF) and interleukin-1 (IL-1) significantly decreased FXR mRNA, whereas IL-6 did not have any effect. TNF and IL-1 also decreased the DNA binding activity to an IR1 response element and the expression of SHP and apoCII. Importantly, TNF and IL-1 almost completely blocked the expression of luciferase activity linked to a FXR response element promoter construct transfected into Hep3B cells. Together with our earlier studies on the repression of RXRs, peroxisome proliferator-activated receptors, LXRs, thyroid receptors, constitutive androstane receptor, and pregnane X receptor, these results suggest that decreases in nuclear hormone receptors are major contributors to the decreased gene expression that occurs in the negative acute phase response. The acute phase response (APR) 1The abbreviations used are: APR, acute phase response; LPS, lipopolysaccharide; TNF, tumor necrosis factor; IL, interleukin; IR, inverted repeat; FXR, farnesoid X receptor; FXRE, FXR response element; RXR, retinoid X receptor; PPAR, peroxisome proliferator-activated receptor; LXR, liver X receptor; SHP, small heterodimer partner; LRH, liver receptor homolog; TR, thyroid receptor; apoCII, apolipoprotein CII; PLTP, phospholipid transfer protein; BSEP, bile salt export pump; CYP7A1, cholesterol 7α-hydroxylase; HNF, hepatocyte nuclear factor; IP, intraperitoneally; CDCA, chenodeoxycholic acid 1The abbreviations used are: APR, acute phase response; LPS, lipopolysaccharide; TNF, tumor necrosis factor; IL, interleukin; IR, inverted repeat; FXR, farnesoid X receptor; FXRE, FXR response element; RXR, retinoid X receptor; PPAR, peroxisome proliferator-activated receptor; LXR, liver X receptor; SHP, small heterodimer partner; LRH, liver receptor homolog; TR, thyroid receptor; apoCII, apolipoprotein CII; PLTP, phospholipid transfer protein; BSEP, bile salt export pump; CYP7A1, cholesterol 7α-hydroxylase; HNF, hepatocyte nuclear factor; IP, intraperitoneally; CDCA, chenodeoxycholic acid is induced during infection, inflammation, and injury and is associated with a wide range of metabolic changes (1Kushner I. Ann. N. Y. Acad. Sci. 1982; 389: 39-48Google Scholar). Among these, changes in lipid metabolism have received much attention due to the link between infection/inflammatory diseases and atherosclerosis (2Libby P. Egan D. Skarlatos S. Circulation. 1997; 96: 4095-4103Google Scholar, 3Jousilahti P. Vartiainen E. Tuomilehto J. Puska P. Lancet. 1996; 348: 567-572Google Scholar, 4Ridker P.M. Hennekens C.H. Buring J.E. Rifai N. N. Engl. J. Med. 2000; 342: 836-843Google Scholar, 5Koenig W. Sund M. Frohlich M. Fischer H.G. Lowel H. Doring A. Hutchinson W.L. Pepys M.B. Circulation. 1999; 99: 237-242Google Scholar, 6Ridker P.M. Buring J.E. Shih J. Matias M. Hennekens C.H. Circulation. 1998; 98: 731-733Google Scholar). The characteristic changes in lipid metabolism during the APR include hypertriglyceridemia (7Feingold K.R. Staprans I. Memon R.A. Moser A.H. Shigenaga J.K. Doerrler W. Dinarello C.A. Grunfeld C. J. Lipid Res. 1992; 33: 1765-1776Google Scholar), decreases in serum high density lipoprotein cholesterol levels (8Sammalkorpi K. Valtonen V. Kerttula Y. Nikkila E. Taskinen M.R. Metabolism. 1988; 37: 859-865Google Scholar, 9Grunfeld C. Pang M. Doerrler W. Shigenaga J.K. Jensen P. Feingold K.R. J. Clin. Endocrinol. Metab. 1992; 74: 1045-1052Google Scholar), increased hepatic cholesterol synthesis, inhibition of bile acid synthesis (10Hardardottir I. Grunfeld C. Feingold K.R. Biochem. Soc. Trans. 1995; 23: 1013-1018Google Scholar), increased hepatic fatty acid synthesis, and decreased hepatic fatty acid oxidation and ketogenesis (11Memon R.A. Feingold K.R. Moser A.H. Doerrler W. Grunfeld C. Endocrinology. 1992; 131: 1695-1702Google Scholar, 12Memon R.A. Feingold K.R. Moser A.H. Doerrler W. Adi S. Dinarello C.A. Grunfeld C. Am. J. Physiol. 1992; 263: E301-E309Google Scholar). These changes are mediated by alterations in gene expression caused by pro-inflammatory cytokines including TNFα, IL-1β, and IL-6 (10Hardardottir I. Grunfeld C. Feingold K.R. Biochem. Soc. Trans. 1995; 23: 1013-1018Google Scholar). However, the underlying mechanism by which these cytokines regulate gene transcription is not well understood, especially for the negative acute phase proteins. Nuclear hormone receptors are ligand-activated transcription factors that are involved in various biological processes including development and physiological homeostasis (13Mangelsdorf D.J. Evans R.M. Cell. 1995; 83: 841-850Google Scholar). Small lipophilic molecules such as steroids, thyroid hormones, vitamin D, and retinoids bind to and activate these receptors to exert their physiological effects by regulating the transcription of specific genes (13Mangelsdorf D.J. Evans R.M. Cell. 1995; 83: 841-850Google Scholar, 14Blumberg B. Evans R.M. Genes Dev. 1998; 12: 3149-3155Google Scholar, 15Kliewer S.A. Lehmann J.M. Willson T.M. Science. 1999; 284: 757-760Google Scholar). These receptors share common structural features, including central, highly conserved DNA binding domains and carboxyl-terminal ligand binding domains (13Mangelsdorf D.J. Evans R.M. Cell. 1995; 83: 841-850Google Scholar, 14Blumberg B. Evans R.M. Genes Dev. 1998; 12: 3149-3155Google Scholar, 15Kliewer S.A. Lehmann J.M. Willson T.M. Science. 1999; 284: 757-760Google Scholar). They can be divided into four major subgroups based on their dimerization and DNA binding properties. Type II receptors are characterized by their DNA binding as a heterodimer with the 9-cis-retinoic acid receptor (RXR) (13Mangelsdorf D.J. Evans R.M. Cell. 1995; 83: 841-850Google Scholar, 16Kliewer S.A. Umesono K. Noonan D.J. Heyman R.A. Evans R.M. Nature. 1992; 358: 771-774Google Scholar). This group includes the peroxisome proliferator-activated receptor (PPAR), retinoic acid receptor, vitamin D receptor, liver X receptor (LXR), and thyroid hormone receptors (TRs) (13Mangelsdorf D.J. Evans R.M. Cell. 1995; 83: 841-850Google Scholar, 17Mangelsdorf D.J. Thummel C. Beato M. Herrlich P. Schutz G. Umesono K. Blumberg B. Kastner P. Mark M. Chambon P. Evans R.M. Cell. 1995; 83: 835-839Google Scholar). The farnesoid X receptor (FXR) was once an orphan receptor, and recently, it was found that bile acids are the ligands for FXR (18Makishima M. Okamoto A.Y. Repa J.J. Tu H. Learned R.M. Luk A. Hull M.V. Lustig K.D. Mangelsdorf D.J. Shan B. Science. 1999; 284: 1362-1365Google Scholar, 19Parks D.J. Blanchard S.G. Bledsoe R.K. Chandra G. Consler T.G. Kliewer S.A. Stimmel J.B. Willson T.M. Zavacki A.M. Moore D.D. Lehmann J.M. Science. 1999; 284: 1365-1368Google Scholar, 20Wang H. Chen J. Hollister K. Sowers L.C. Forman B.M. Mol. Cell. 1999; 3: 543-553Google Scholar). FXR forms an obligate heterodimer with RXR and, thus, belongs to the Type II nuclear receptor subgroup. FXR has been shown to bind to FXR response elements (FXRE) composed of two inverted hexanucleotide repeats (AGGTCA) spaced by one nucleotide (IR-1) (21Laffitte B.A. Kast H.R. Nguyen C.M. Zavacki A.M. Moore D.D. Edwards P.A. J. Biol. Chem. 2000; 275: 10638-10647Google Scholar). Chenodeoxycholic acid (CDCA), the most potent ligand for FXR, induces the ileal bile acid-binding protein (I-BABP) (18Makishima M. Okamoto A.Y. Repa J.J. Tu H. Learned R.M. Luk A. Hull M.V. Lustig K.D. Mangelsdorf D.J. Shan B. Science. 1999; 284: 1362-1365Google Scholar, 22Kanda T. Foucand L. Nakamura Y. Niot I. Besnard P. Fujita M. Sakai Y. Hatakeyama K. Ono T. Fujii H. Biochem. J. 1998; 330: 261-265Google Scholar), bile salt export pump (23Ananthanarayanan M. Balasubramanian N. Makishima M. Mangelsdorf D.J. Suchy F.J. J. Biol. Chem. 2001; 276: 28857-28865Google Scholar), phospholipid transfer protein (PLTP) (21Laffitte B.A. Kast H.R. Nguyen C.M. Zavacki A.M. Moore D.D. Edwards P.A. J. Biol. Chem. 2000; 275: 10638-10647Google Scholar, 24Urizar N.L. Dowhan D.H. Moore D.D. J. Biol. Chem. 2000; 275: 39313-39317Google Scholar), apolipoprotein CII (apoCII) (25Kast H.R. Nguyen C.M. Sinal C.J. Jones S.A. Laffitte B.A. Reue K. Gonzalez F.J. Willson T.M. Edwards P.A. Mol. Endocrinol. 2001; 15: 1720-1728Google Scholar), and SHP (26Goodwin B. Jones S.A. Price R.R. Watson M.A. McKee D.D. Moore L.B. Galardi C. Wilson J.G. Lewis M.C. Roth M.E. Maloney P.R. Willson T.M. Kliewer S.A. Mol. Cell. 2000; 6: 517-526Google Scholar). Activation of FXR down-regulates the expression of CYP7A1 via the action of SHP protein (26Goodwin B. Jones S.A. Price R.R. Watson M.A. McKee D.D. Moore L.B. Galardi C. Wilson J.G. Lewis M.C. Roth M.E. Maloney P.R. Willson T.M. Kliewer S.A. Mol. Cell. 2000; 6: 517-526Google Scholar). FXR-induced SHP binds to and inactivates the liver receptor homolog 1(LRH), a transcription factor that is required for the expression of cholesterol 7α-hydroxylase (CYP7A1) (26Goodwin B. Jones S.A. Price R.R. Watson M.A. McKee D.D. Moore L.B. Galardi C. Wilson J.G. Lewis M.C. Roth M.E. Maloney P.R. Willson T.M. Kliewer S.A. Mol. Cell. 2000; 6: 517-526Google Scholar, 27Lu T.T. Makishima M. Repa J.J. Schoonjans K. Kerr T.A. Auwerx J. Mangelsdorf D.J. Mol. Cell. 2000; 6: 507-515Google Scholar). CYP7A1 is a rate-limiting enzyme in the neutral pathway of bile acid synthesis (28Chiang J.Y.L. Front Biosci. 1998; 3: 176-193Google Scholar), and therefore, FXR activation inhibits bile acid biosynthesis. Because bile acid is synthesized from cholesterol in the liver and is the major route for the elimination of cholesterol from the body, regulation of CYP7A1 transcription and activity indicates a critical role of FXR in the regulation of cholesterol metabolism. A number of genes involved in lipid metabolism whose hepatic expression is decreased during the acute phase response are known to be regulated by type II nuclear hormone receptors. Previously, we demonstrated that in Syrian hamsters, LPS administration results in a decrease in hepatic mRNA and/or protein levels of RXRα, -β, and -γ, PPARα and -γ, TRα and -β, and LXRα (29Beigneux A.P. Moser A.H. Shigenaga J.K. Grunfeld C. Feingold K.R. J. Biol. Chem. 2000; 275: 16390-16399Google Scholar, 30Beigneux A.P. Moser A.H. Shigenaga J.K. Grunfeld C. Feingold K.R. Am. J. Physiol. Endocrinol. Metab. 2003; 284: E228-E236Google Scholar). Furthermore, hepatic nuclear extracts obtained from animals treated with LPS exhibited a reduced binding activity to RXR-RXR, RXR-PPAR, RXR-TR, and RXR-LXR response elements (29Beigneux A.P. Moser A.H. Shigenaga J.K. Grunfeld C. Feingold K.R. J. Biol. Chem. 2000; 275: 16390-16399Google Scholar, 30Beigneux A.P. Moser A.H. Shigenaga J.K. Grunfeld C. Feingold K.R. Am. J. Physiol. Endocrinol. Metab. 2003; 284: E228-E236Google Scholar). This suggests that reduced hepatic RXR levels alone or in combination with decreases in PPARs and LXR could be a mechanism for coordinately inhibiting the expression of multiple genes during the acute phase response. Expression of two proteins that are regulated by FXR, bile salt export pump (BSEP) (31Hartmann G. Cheung A.K. Piquette-Miller M. J. Pharmacol. Exp. Ther. 2002; 303: 273-281Google Scholar) and phospholipid transfer protein (PLTP) (32Jiang X.C. Bruce C. J. Biol. Chem. 1995; 270: 17133-17138Google Scholar), are decreased during the APR. Therefore, we hypothesized that the bile acid receptor FXR is also suppressed during APR, affecting lipid/cholesterol metabolism. In the present study, we demonstrate that LPS and pro-inflammatory cytokines TNFα and IL-1β decrease the expression of FXR, its DNA binding activity to FXR response element (IR1), and transcription by FXR in studies of mice in vivo and the Hep3B human hepatoma cells in vitro, which is accompanied by decreased expression of FXR-regulated genes, SHP, and apoCII. The data suggest that altered FXR activity may contribute to the changes in lipid and cholesterol metabolism that occur during the APR. LPS (Escherichia coli 55:B5) was obtained from Difco and freshly diluted to the desired concentration in pyrogen-free 0.9% saline. Minimum essential medium (MEM) was purchased from Fisher. Cytokines (human TNFα, human IL-1β, and human IL-6) were from R&D Systems and were freshly diluted to desired concentrations in serum-free MEM media containing 0.1% bovine serum albumin (fatty acid-free). Tri-Reagent and fatty acid-free bovine serum albumin were from Sigma. [α-32P]dCTP (3,000 Ci/mmol) and [γ-32P]dATP (3,000 Ci/mmol) were purchased from PerkinElmer Life Sciences. Oligo(dT)-cellulose type 77F was fromAmersham Biosciences. Eight-week-old female C57BL/6 mice were purchased from The Jackson Laboratory (Bar Harbor, ME). The animals were maintained in a normal light-cycle room and were provided with rodent chow and water ad libitum. Anesthesia was induced with halothane. To determine the effect of APR on FXR and other mRNA levels, mice were injected IP with 100 μg of LPS in saline or with saline alone. Food was withdrawn at the time of injection, because LPS induces anorexia in rodents (33Grunfeld C. Zhao C. Fuller J. Pollack A. Moser A. Friedman J. Feingold K.R. J. Clin. Invest. 1996; 97: 2152-2157Google Scholar). Livers were removed at the time indicated in the figure legends (Figs. 1, 2, 4) after treatment. The doses of LPS used in this study have significant effects on triglyceride and cholesterol metabolism (7Feingold K.R. Staprans I. Memon R.A. Moser A.H. Shigenaga J.K. Doerrler W. Dinarello C.A. Grunfeld C. J. Lipid Res. 1992; 33: 1765-1776Google Scholar) but are not lethal because the LD50 for LPS in rodents is ∼5 mg/100 g of body weight.Figure 2Effect of LPS treatment on binding of hepatic nuclear extracts to FXR response element IR1. C57BL/6 mice were injected IP with either saline or LPS (100 μg of LPS/mouse). Sixteen hours later, the animals were sacrificed, and hepatic nuclear extracts (NE) were prepared as described under “Experimental Procedures.” Ten micrograms of nuclear extracts were incubated with radiolabeled oligonucleotides IR1 representing binding site for FXR.A, a representative electrophoretic gel mobility shift assay. Unlabeled specific (100× WT) and nonspecific (100× Mut) competing oligonucleotides were included at a 100-fold excess 1 h before the addition of the labeled probes.Arrows represent specific IR1-bound complexes. B, densitometric analysis of hepatic DNA-binding proteins. Data (means ± S.E., n = 5) are expressed as a percentage of controls. *, p < 0.05 versuscontrol.View Large Image Figure ViewerDownload (PPT)Figure 4Effect of LPS on the expression of FXR target genes SHP and apoCII. Hepatic total RNA was prepared 16 h after saline or LPS administration (100 μg of LPS/mouse) from mouse liver. Northern blot analysis was performed as described under “Experimental Procedures” using SHP and apoCII cDNAs. Data (means ± S.E., n = 4∼5) are expressed as a percentage of controls. *, p < 0.05 versuscontrol.View Large Image Figure ViewerDownload (PPT) Hep3B cells were maintained in MEM medium supplemented with 10% fetal bovine serum in 75-cm2 flasks. Cells were washed twice with phosphate-buffered saline (Ca2+- and Mg2+-free) and trypsinized before seeding. For typical experiments, cells were seeded in 100-mm dishes at a concentration of 2 × 106 cells/dish. After an overnight incubation, cells were washed twice with phosphate-buffered saline, and medium was replaced with fresh MEM (without serum) plus 0.1% bovine albumin and the appropriate cytokine concentration. For transfection assays, 1.5 × 105 cells were used/well in 6-well plates. Total RNA from mouse was isolated from 300–400 mg of snap-frozen whole liver and ∼100 mg of kidney tissue using Tri-Reagent (Sigma). Poly(A)+ RNA was subsequently purified using oligo(dT) cellulose. RNA was quantified by measuring absorption at 260 nm. 10 μg of poly(A)+ were denatured and electrophoresed on a 1% agarose, formaldehyde gel. Total RNA from Hep3B was isolated from a 100-mm dish by the Tri-Reagent method and resuspended in diethyl pyrocarbonate-treated water. 30 μg of total RNA was denatured and electrophoresed as described above. The uniformity of sample loading was checked by UV visualization of the ethidium bromide-stained gel before electrotransfer to Nytran membrane (Schleicher & Schuell). Prehybridization, hybridization, and washing procedures were performed as described previously (34Memon R.A. Fuller J. Moser A.H. Smith P.J. Feingold K.R. Grunfeld C. Am. J. Physiol. 1998; 275: E64-E72Google Scholar). Membranes were probed with [α-32P]dCTP-labeled cDNAs using the random priming technique (Amersham Biosciences). mRNA levels were detected by exposure of the membrane to x-ray film and quantified by densitometry. Glyceraldehyde-3-phosphate dehydrogenase was used as a control probe. LRH-1 cDNA was kindly provided by Dr. Kristina Schoonjans (Institut de Genetique et de Moleculaire et Cellulaire, Universite Louis Pasteur, Paris, France). Mouse and human FXR, SHP, and apoCII probes were prepared by PCR using the following primers: FXR 5′-CGT GAC TTG CGN CAA GTG ACC-3′ (upper), 5′-CCA NGA CAT CAG CAT CTC AGC-3′ (lower); SHP 5′-AGG GGT CTG CCC ATG CCA G-3′ (upper), 5′-GGT CAC CTC AGC AAA AGC ATG TC-3′ (lower); apoCII 5′-GCC AAG GAG GTT GCC AAA G-3′ (upper), 5′-GGT CTG GTG ATG CGA GCA A-3′ (lower). Nuclear extracts were prepared according to Neish et al. (35Neish A.S. Khachigian L.M. Park A. Baichwal V.R. Collins T. J. Biol. Chem. 1995; 270: 28903-28909Google Scholar). Briefly, cells were disrupted in a sucrose-HEPES buffer containing 0.5% Nonidet-P40 as a detergent, protease inhibitors, and dithiothreitol. After disruption by 5 min of incubation on ice and centrifugation, nuclear proteins were separated in a NaCl-HEPES buffer and re-suspended in a glycerol-containing buffer. All the procedures were carried out on ice. Protein quantification was determined by the Bradford assay (Bio-Rad), and yields were similar in control and cytokine-treated groups. 10 μg of crude nuclear extract were incubated on ice for 30 min with 6 × 104cpm of 32P-labeled oligonucleotides in 15 μl of binding buffer consisting of 20% glycerol, 25 mm Tris-HCl, pH 7.5, 40 mm KCl, 0.5 mm MgCl2, 0.1 mm EDTA, 1 mm dithiothreitol, 2 μg of poly(dI-dC), and 1 μg of salmon sperm DNA. Double-stranded oligonucleotide probes were end-labeled with T4-polynucleotide kinase (Amersham Biosciences) in the presence of 50 μCi of [γ-32P]dATP and purified on a Sephadex G-25 column (Amersham Biosciences). DNA-protein complexes were separated by electrophoresis (constant voltage of 200 V) on a 5% nondenaturing polyacrylamide gel in 0.5× Tris-buffered EDTA at 4 °C. The gel was dried, exposed to x-ray film, and quantified by densitometry. In the competition assay, a 100-fold molar excess of the specific or mutated unlabeled oligonucleotide was preincubated on ice for 1 h with 10 μg of nuclear extract from control cells. The following oligonucleotides were used: IR1, 5′-GATCGGCCAGGGTGAATAACCTCGGGG-3′; mut-IR1, 5′-GATCGGCCAGGAAGAATATTCTCGGGG-3′. Hep3B cells were grown overnight in 35-mm plates and washed twice with serum-free medium. DNA-Lipofectin complex containing 1.5 μg/ml FXRE-luciferase vector (a gift from Dr. Mangelsdorf, University of Texas Southwestern Medical Center, Dallas, TX), 0.5 μg/ml Rous sarcoma virus β-galactosidase vector (a gift from Dr. Allan Pollock, Nephrology section at Veterans Affairs Medical Center, San Francisco, CA), and 5 μg/ml Lipofectin (Invitrogen) were allowed to form at room temperature for 15 min. The cells were overlaid with the DNA-Lipofectin complex and incubated at 37 °C for 4–6 h. After washing the cells with serum-free medium, fresh growth medium containing 10% fetal bovine serum was added. Transfected cells were treated with lysis buffer (Promega, Madison, WI), and aliquots of the lysates were assayed for luciferase and β-galactosidase enzyme activity as described in the manufacturer's instruction using Wallac VICTOR2™ 1420 Multilabel Counter (PerkinElmer Life Sciences). β-Galactosidase enzyme activity was used to normalize for variability in the transfection efficiency. Data are expressed as the mean ±S.E. of experiments from 3–5 animals or plates for each groups or time point. The difference between two experimental groups was analyzed using the Student's t test. Differences among multiple groups were analyzed using one way analysis of variance with the Bonferroni's post hoc. A p value < 0.05 was considered significant. We initially determined the effect of LPS administration on the FXR mRNA levels in mouse liver at various times up to 24 h. As shown in Fig.1 A, LPS (100 μg) administration did not cause any significant change in the FXR mRNA level at 2 h. However, FXR mRNA decreased significantly by 8 h to ∼40% that of the control level, and the reduction persisted for 24 h. Next, we determined if FXR mRNA decreases in a dose-dependent manner in response to LPS administration. As shown in Fig. 1 B, the LPS-induced decrease in FXR mRNA was dose-dependent in mouse liver, with the half-maximal effect occurring at ∼0.5 μg/100 g of body weight. All doses tested in the experiment caused a significant reduction in FXR mRNA levels. Thus, LPS decreases FXR mRNA levels in the liver of mice at relatively low doses. Because hepatic expression of FXR is down-regulated by LPS administration in mouse liver, next we examined if FXR expression is altered in the intestine and the kidney, which also express FXR. As in the liver, LPS administration for 16 h caused a significant decrease (∼55%) in the level of FXR mRNA in the kidney (Fig.1 C). However, FXR mRNA was not reduced in the intestine (∼130%), suggesting that LPS causes tissue-specific responses in terms of FXR expression. These results indicate that LPS-induced APR reduces expression of FXR in mouse liver and kidney but not in the intestine. Nuclear hormone receptors exert their effect on transcriptional regulation by binding to their cognitive response element in the promoter region of target genes. To determine whether the reduction of FXR mRNA caused by LPS administration affects the DNA binding activity of FXR, we isolated nuclei from mouse liver and performed the electrophoretic gel mobility shift assay using a32P-labeled DNA oligonucleotide containing FXR response element IR-1. As shown in Fig.2 A, two major FXR·IR1 complexes were observed in the control samples. LPS administration significantly decreased the binding of proteins in the nuclear extract from mouse liver to IR1 when compared with the control. LPS decreased FXR·IR1 binding by ∼75 and ∼50% for the upper and the lower band, respectively (Fig. 2 B). Competition with 100-fold molar excess of specific oligonucleotide (WT), but not with mutated oligonucleotide (Mut), abolished the complex formation of radiolabeled IR1 with FXR (Fig. 2 A), demonstrating the specificity of the two complexes. Furthermore, we demonstrated that the complexes contain FXR and RXR. They were supershifted with anti-FXR (SS1, second lane) and anti-RXR (SS2, third lane) antibody (Fig.3). The migration of FXR·DNA complex was not affected by nonspecific IgG (fifth lane). To determine whether the decreased binding of FXR to DNA in hepatic nuclear extracts from LPS-treated mouse is associated with reduced transcription of FXR-regulated genes, we investigated the effect of LPS administration on the mRNA levels of SHP and apoCII, two of genes that are known to be regulated by FXR. SHP is a key mediator of the FXR effect on the transcriptional regulation of CYP7A1 gene, and SHP is induced by FXR activation. As shown in Fig.4, LPS administration decreases the expression of SHP by ∼70% and apoCII by 60% in mouse liver when compared with control. These data demonstrate that FXR-regulated genes are also repressed during LPS-induced APR in the mouse. It is well established that FXR down-regulates CYP7A1 expression as a feedback mechanism to maintain homeostasis of bile acid metabolism (26Goodwin B. Jones S.A. Price R.R. Watson M.A. McKee D.D. Moore L.B. Galardi C. Wilson J.G. Lewis M.C. Roth M.E. Maloney P.R. Willson T.M. Kliewer S.A. Mol. Cell. 2000; 6: 517-526Google Scholar, 27Lu T.T. Makishima M. Repa J.J. Schoonjans K. Kerr T.A. Auwerx J. Mangelsdorf D.J. Mol. Cell. 2000; 6: 507-515Google Scholar). This is achieved by FXR-induced stimulation of SHP expression (26Goodwin B. Jones S.A. Price R.R. Watson M.A. McKee D.D. Moore L.B. Galardi C. Wilson J.G. Lewis M.C. Roth M.E. Maloney P.R. Willson T.M. Kliewer S.A. Mol. Cell. 2000; 6: 517-526Google Scholar). SHP binds to and inactivates LRH, which is required for the transcriptional activation of CYP7A1 (26Goodwin B. Jones S.A. Price R.R. Watson M.A. McKee D.D. Moore L.B. Galardi C. Wilson J.G. Lewis M.C. Roth M.E. Maloney P.R. Willson T.M. Kliewer S.A. Mol. Cell. 2000; 6: 517-526Google Scholar). Therefore, based on the above results showing a decrease in FXR and SHP, it is reasonable to expect an increase in the level of CYP7A1 during the APR. However, our previous studies demonstrated that the expression of Cyp7A1 is down-regulated during the APR induced by LPS administration in vivo (36Feingold K.R. Spady D.K. Pollock A.S. Moser A.H. Grunfeld C. J. Lipid Res. 1996; 37: 223-228Google Scholar). Thus, we determined the effect of LPS on the expression of LRH in mouse liver. As shown in Fig. 5, LPS administration for 16 h caused an ∼65% reduction in the LRH mRNA levels in mouse liver. This result suggests that the LPS-induced decrease in LRH may play a major role in determining the transcriptional regulation of CYP7A1. It is well known that the physiological effect of LPS is mediated by pro-inflammatory cytokines such as TNF, IL-1, and IL-6. To determine whether these cytokines also decrease FXR mRNAin vitro, as LPS does in mouse liver, human hepatoma cell line Hep3B cells were treated with cytokines at 10 ng/ml for 24 h, and RNA was isolated for Northern analysis. As shown in Fig.6, TNF treatment decreased FXR mRNA to less than 20% that of the control level. Also, IL-1 decreased FXR mRNA by 60%. However, IL-6 did not affect FXR expression in Hep3B cells (data not shown). These results suggest that the effect of LPS on FXR expression is mediated by TNF and IL-1. Because the inflammatory cytokine TNF causes a reduction in the FXR mRNA, we next determined if TNF also causes a similar change in the DNA binding activity of nuclear extracts from human hepatoma cells to an IR-1 FXR response element. For this experiment, Hep3" @default.
• W2071489706 created "2016-06-24" @default.
• W2071489706 creator A5037226938 @default.
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• W2071489706 date "2003-03-01" @default.
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• W2071489706 title "Repression of Farnesoid X Receptor during the Acute Phase Response" @default.
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• W2071489706 doi "https://doi.org/10.1074/jbc.m212633200" @default.
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