Regulation of Bile Acid Synthesis by the Nuclear Receptor Rev-erbα
2008; Elsevier BV; Volume: 135; Issue: 2 Linguagem: Inglês
10.1053/j.gastro.2008.05.035
ISSN1528-0012
AutoresHélène Duez, Jelske N. van der Veen, Christian Duhem, Benoît Pourcet, Thierry Touvier, Coralie Fontaine, Bruno Derudas, Eric Baugé, Rick Havinga, Vincent W. Bloks, Henk Wolters, Fjodor H. van der Sluijs, Björn Vennström, Folkert Kuipers, Bart Staels,
Tópico(s)Pharmacogenetics and Drug Metabolism
ResumoBackground & Aims: Conversion into bile acids represents an important route to remove excess cholesterol from the body. Rev-erbα is a nuclear receptor that participates as one of the clock genes in the control of circadian rhythmicity and plays a regulatory role in lipid metabolism and adipogenesis. Here, we investigate a potential role for Rev-erbα in the control of bile acid metabolism via the regulation of the neutral bile acid synthesis pathway. Methods: Bile acid synthesis and CYP7A1 gene expression were studied in vitro and in vivo in mice deficient for or over expressing Rev-erbα. Results: Rev-erbα-deficient mice display a lower synthesis rate and an impaired excretion of bile acids into the bile and feces. Expression of CYP7A1, the rate-limiting enzyme of the neutral pathway, is decreased in livers of Rev-erbα-deficient mice, whereas adenovirus-mediated hepatic Rev-erbα overexpression induces its expression. Moreover, bile acid feeding resulted in a more pronounced suppression of hepatic CYP7A1 expression in Rev-erbα-deficient mice. Hepatic expression of E4BP4 and the orphan nuclear receptor small heterodimer partner (SHP), both negative regulators of CYP7A1 expression, is increased in Rev-erbα-deficient mice. Promoter analysis and chromatin immunoprecipitation experiments demonstrated that SHP and E4BP4 are direct Rev-erbα target genes. Finally, the circadian rhythms of liver CYP7A1, SHP, and E4BP4 messenger RNA levels were perturbed in Rev-erbα-deficient mice. Conclusions: These data identify a role for Rev-erbα in the regulatory loop of bile acid synthesis, likely acting by regulating both hepatic SHP and E4BP4 expression. Background & Aims: Conversion into bile acids represents an important route to remove excess cholesterol from the body. Rev-erbα is a nuclear receptor that participates as one of the clock genes in the control of circadian rhythmicity and plays a regulatory role in lipid metabolism and adipogenesis. Here, we investigate a potential role for Rev-erbα in the control of bile acid metabolism via the regulation of the neutral bile acid synthesis pathway. Methods: Bile acid synthesis and CYP7A1 gene expression were studied in vitro and in vivo in mice deficient for or over expressing Rev-erbα. Results: Rev-erbα-deficient mice display a lower synthesis rate and an impaired excretion of bile acids into the bile and feces. Expression of CYP7A1, the rate-limiting enzyme of the neutral pathway, is decreased in livers of Rev-erbα-deficient mice, whereas adenovirus-mediated hepatic Rev-erbα overexpression induces its expression. Moreover, bile acid feeding resulted in a more pronounced suppression of hepatic CYP7A1 expression in Rev-erbα-deficient mice. Hepatic expression of E4BP4 and the orphan nuclear receptor small heterodimer partner (SHP), both negative regulators of CYP7A1 expression, is increased in Rev-erbα-deficient mice. Promoter analysis and chromatin immunoprecipitation experiments demonstrated that SHP and E4BP4 are direct Rev-erbα target genes. Finally, the circadian rhythms of liver CYP7A1, SHP, and E4BP4 messenger RNA levels were perturbed in Rev-erbα-deficient mice. Conclusions: These data identify a role for Rev-erbα in the regulatory loop of bile acid synthesis, likely acting by regulating both hepatic SHP and E4BP4 expression. Conversion of cholesterol into bile acids occurs via a complex biosynthetic pathway involving up to 17 enzymatic steps.1Russell D.W. The enzymes, regulation, and genetics of bile acid synthesis.Annu Rev Biochem. 2003; 72: 137-174Crossref PubMed Scopus (1467) Google Scholar Subsequently, bile acids are stored in the gallbladder and expelled into the intestinal lumen upon ingestion of a meal. Bile acids exert a number of physiologic functions, participating in the excretion of hepatic cholesterol into bile, in the generation of bile flow, and in intestinal solubilization and absorption of fat. Bile acids also have important regulatory functions in lipid and glucose metabolism.2Kuipers F. Stroeve J.H. Caron S. et al.Bile acids, farnesoid X receptor, atherosclerosis and metabolic control.Curr Opin Lipidol. 2007; 18: 289-297Crossref PubMed Scopus (58) Google Scholar, 3Sinal C.J. Tohkin M. Miyata M. et al.Targeted disruption of the nuclear receptor FXR/BAR impairs bile acid and lipid homeostasis.Cell. 2000; 102: 731-744Abstract Full Text Full Text PDF PubMed Scopus (1478) Google Scholar, 4Watanabe M. Houten S.M. Wang L. et al.Bile acids lower triglyceride levels via a pathway involving FXR, SHP, and SREBP-1c.J Clin Invest. 2004; 113: 1408-1418Crossref PubMed Scopus (1022) Google Scholar, 5Angelin B. Eriksson M. Rudling M. Bile acids and lipoprotein metabolism: a renaissance for bile acids in the post-statin era?.Curr Opin Lipidol. 1999; 10: 269-274Crossref PubMed Scopus (22) Google Scholar, 6Kast H.R. Nguyen C.M. Sinal C.J. et al.Farnesoid X-activated receptor induces apolipoprotein C-II transcription: a molecular mechanism linking plasma triglyceride levels to bile acids.Mol Endocrinol. 2001; 15: 1720-1728Crossref PubMed Scopus (0) Google Scholar From the intestinal lumen, bile acids are reabsorbed and delivered back to the liver (enterohepatic circulation) via the concerted action of several transporters. A small fraction of bile acids escapes absorption and is lost via excretion in the feces.7Kuipers F. Claudel T. Sturm E. et al.The farnesoid X receptor (FXR) as modulator of bile acid metabolism.Rev Endocr Metab Disord. 2004; 5: 319-326Crossref PubMed Scopus (57) Google Scholar Under steady-state conditions, this fecal loss is compensated for by de novo synthesis in the liver to maintain the size of the circulating bile acid pool. Bile acids exert a negative feedback control on their own synthesis by down-regulating the expression of the cholesterol 7α-hydroxylase (CYP7A1) gene, which encodes the first and rate-controlling enzyme of the major bile acid biosynthetic pathway,1Russell D.W. The enzymes, regulation, and genetics of bile acid synthesis.Annu Rev Biochem. 2003; 72: 137-174Crossref PubMed Scopus (1467) Google Scholar, 8Russell D.W. Setchell K.D. Bile acid biosynthesis.Biochemistry. 1992; 31: 4737-4749Crossref PubMed Scopus (673) Google Scholar via a mechanism implicating the bile acid-activated nuclear receptor farnesoid X receptor (FXR, NR1H4). FXR indirectly inhibits CYP7A1 expression by both inducing expression of fibroblast growth factor-15 in the intestine9Inagaki T. Choi M. Moschetta A. et al.Fibroblast growth factor 15 functions as an enterohepatic signal to regulate bile acid homeostasis.Cell Metab. 2005; 2: 217-225Abstract Full Text Full Text PDF PubMed Scopus (1389) Google Scholar, 10Li J. Pircher P.C. Schulman I.G. et al.Regulation of complement C3 expression by the bile acid receptor FXR.J Biol Chem. 2005; 280: 7427-7434Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar as well as small heterodimer partner (SHP) in the liver.11Lu T.T. Makishima M. Repa J.J. et al.Molecular basis for feedback regulation of bile acid synthesis by nuclear receptors.Mol Cell. 2000; 6: 507-515Abstract Full Text Full Text PDF PubMed Scopus (1242) Google Scholar, 12Goodwin B. Jones S.A. Price R.R. et al.A regulatory cascade of the nuclear receptors FXR, SHP-1, and LRH-1 represses bile acid biosynthesis.Mol Cell. 2000; 6: 517-526Abstract Full Text Full Text PDF PubMed Scopus (1551) Google Scholar In addition, bile acids also down-regulate their synthesis either via activation of other nuclear receptors, such as the pregnane X receptor (PXR, NR1I2),13Staudinger J.L. Goodwin B. Jones S.A. et al.The nuclear receptor PXR is a lithocholic acid sensor that protects against liver toxicity.Proc Natl Acad Sci U S A. 2001; 98: 3369-3374Crossref PubMed Scopus (1167) Google Scholar or via the activation of several kinase-dependent signalling pathways.14Gupta S. Stravitz R.T. Dent P. et al.Down-regulation of cholesterol 7α-hydroxylase (CYP7A1) gene expression by bile acids in primary rat hepatocytes is mediated by the c-Jun N-terminal kinase pathway.J Biol Chem. 2001; 276: 15816-15822Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar, 15Wang L. Lee Y.K. Bundman D. et al.Redundant pathways for negative feedback regulation of bile acid production.Dev Cell. 2002; 2: 721-731Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar, 16Holt J.A. Luo G. Billin A.N. et al.Definition of a novel growth factor-dependent signal cascade for the suppression of bile acid biosynthesis.Genes Dev. 2003; 17: 1581-1591Crossref PubMed Scopus (548) Google Scholar Moreover, CYP7A1 expression is induced by liver X receptor (LXR, NR1H3) in mice17Peet D.J. Turley S.D. Ma W. et al.Cholesterol and bile acid metabolism are impaired in mice lacking the nuclear oxysterol receptor LXR α.Cell. 1998; 93: 693-704Abstract Full Text Full Text PDF PubMed Scopus (1263) Google Scholar as well as by the hepatic nuclear factor (HNF) 4α.18Hayhurst G.P. Lee Y.H. Lambert G. et al.Hepatocyte nuclear factor 4α (nuclear receptor 2A1) is essential for maintenance of hepatic gene expression and lipid homeostasis.Mol Cell Biol. 2001; 21: 1393-1403Crossref PubMed Scopus (880) Google Scholar CYP7A1 messenger RNA (mRNA) levels and activity show strong diurnal fluctuations19Sundseth S.S. Waxman D.J. Hepatic P-450 cholesterol 7α-hydroxylase Regulation in vivo at the protein and mRNA level in response to mevalonate, diurnal rhythm, and bile acid feedback.J Biol Chem. 1990; 265: 15090-15095Abstract Full Text PDF PubMed Google Scholar, 20Kai M. Eto T. Kondo K. et al.Synchronous circadian rhythms of mRNA levels and activities of cholesterol 7α-hydroxylase in the rabbit and rat.J Lipid Res. 1995; 36: 367-374Abstract Full Text PDF PubMed Google Scholar, 21Li Y.C. Wang D.P. Chiang J.Y. Regulation of cholesterol 7α-hydroxylase in the liver Cloning, sequencing, and regulation of cholesterol 7α-hydroxylase mRNA.J Biol Chem. 1990; 265: 12012-12019Abstract Full Text PDF PubMed Google Scholar, 22Noshiro M. Nishimoto M. Okuda K. Rat liver cholesterol 7α-hydroxylase Pretranslational regulation for circadian rhythm.J Biol Chem. 1990; 265: 10036-10041Abstract Full Text PDF PubMed Google Scholar, 23Panda S. Antoch M.P. Miller B.H. et al.Coordinated transcription of key pathways in the mouse by the circadian clock.Cell. 2002; 109: 307-320Abstract Full Text Full Text PDF PubMed Scopus (1918) Google Scholar because CYP7A1 expression is under the control of the clock genes D-site binding protein (DBP),24Lavery D.J. Schibler U. Circadian transcription of the cholesterol 7α-hydroxylase gene may involve the liver-enriched bZIP protein DBP.Genes Dev. 1993; 7: 1871-1884Crossref PubMed Scopus (266) Google Scholar Dec2,25Noshiro M. Kawamoto T. Furukawa M. et al.Rhythmic expression of DEC1 and DEC2 in peripheral tissues:DEC2 is a potent suppressor for hepatic cytochrome P450s opposing DBP.Genes Cells. 2004; 9: 317-329Crossref PubMed Scopus (61) Google Scholar and E4BP4.26Noshiro M. Usui E. Kawamoto T. et al.Multiple mechanisms regulate circadian expression of the gene for cholesterol 7α-hydroxylase (Cyp7a), a key enzyme in hepatic bile acid biosynthesis.J Biol Rhythms. 2007; 22: 299-311Crossref PubMed Scopus (77) Google Scholar Rev-erbα (NR1D1) is a nuclear receptor that behaves as a transcriptional repressor by binding either as a monomer or as a homodimer to specific response elements located in the promoters of its target genes.27Harding H.P. Lazar M.A. The monomer-binding orphan receptor Rev-Erb represses transcription as a dimer on a novel direct repeat.Mol Cell Biol. 1995; 15: 4791-4802Crossref PubMed Scopus (177) Google Scholar Rev-erbα has long been referred to as an “orphan” receptor. Very recently, heme has been identified as a physiologic ligand.28Yin L. Wu N. Curtin J.C. et al.Rev-erbα, a heme sensor that coordinates metabolic and circadian pathways.Science. 2007; 318: 1786-1789Crossref PubMed Scopus (580) Google Scholar, 29Raghuram S. Stayrook K.R. Huang P. et al.Identification of heme as the ligand for the orphan nuclear receptors REV-ERBα and REV-ERBβ.Nat Struct Mol Biol. 2007; 14: 1207-1213Crossref PubMed Scopus (453) Google Scholar Rev-erbα is involved in the control of lipid30Vu-Dac N. Chopin-Delannoy S. Gervois P. et al.The nuclear receptors peroxisome proliferator-activated receptor α and Rev-erbα mediate the species-specific regulation of apolipoprotein A-I expression by fibrates.J Biol Chem. 1998; 273: 25713-25720Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar, 31Raspe E. Duez H. Mansen A. et al.Identification of Rev-erbα as a physiological repressor of apoC-III gene transcription.J Lipid Res. 2002; 43: 2172-2179Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar and glucose28Yin L. Wu N. Curtin J.C. et al.Rev-erbα, a heme sensor that coordinates metabolic and circadian pathways.Science. 2007; 318: 1786-1789Crossref PubMed Scopus (580) Google Scholar metabolism, adipocyte differentiation,32Fontaine C. Dubois G. Duguay Y. et al.The orphan nuclear receptor Rev-Erbα is a peroxisome proliferator-activated receptor (PPAR) γ target gene and promotes PPARγ-induced adipocyte differentiation.J Biol Chem. 2003; 278: 37672-37680Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 33Wang J. Lazar M.A. Bifunctional role of Rev-erbα in adipocyte differentiation.Mol Cell Biol. 2008; 28: 2213-2220Crossref PubMed Scopus (95) Google Scholar muscle physiology,34Pircher P. Chomez P. Yu F. et al.Aberrant expression of myosin isoforms in skeletal muscles from mice lacking the rev-erbAalpha orphan receptor gene.Am J Physiol Regul Integr Comp Physiol. 2005; 288: R482-R490Crossref PubMed Scopus (35) Google Scholar and brain development.35Chomez P. Neveu I. Mansen A. et al.Increased cell death and delayed development in the cerebellum of mice lacking the rev-erbA(α) orphan receptor.Development. 2000; 127: 1489-1498Crossref PubMed Google Scholar Rev-erbα also plays a key role in the regulation of circadian rhythms.36Preitner N. Damiola F. Lopez-Molina L. et al.The orphan nuclear receptor REV-ERBZα controls circadian transcription within the positive limb of the mammalian circadian oscillator.Cell. 2002; 110: 251-260Abstract Full Text Full Text PDF PubMed Scopus (1718) Google Scholar In this study, we demonstrate that Rev-erbα modulates in vivo bile acid metabolism via derepression of CYP7A1 through down-regulation of hepatic SHP and E4BP4 expression. Rev-erbα-deficient mice were generated as previously described35Chomez P. Neveu I. Mansen A. et al.Increased cell death and delayed development in the cerebellum of mice lacking the rev-erbA(α) orphan receptor.Development. 2000; 127: 1489-1498Crossref PubMed Google Scholar and backcrossed for 6 generations into the SV129/OlaHsd genetic background in our animal facility. Rev-erbα-deficient mice and their wild-type littermates were used. Mice were housed in a light/dark cycle (light on at 8:00 am, off at 8:00 pm) and were allowed ad libitum access to food and water. They received a regular chow diet (A03; Usine d'Alimentation Rationelle, France) unless indicated otherwise. All mice were killed in the afternoon (approximately 4 pm) or around the clock every 6 hours. Mice received taurocholic acid (TCA) (Sigma–Aldrich, L'Isle d'Abeau, France) mixed in the regular diet (0.5% wt/wt) for 5 days. Animal care and experimental procedures were performed according to approved institutional guidelines. Mice were subjected to bile duct cannulation for 30 minutes as previously described.37Kuipers F. van Ree J.M. Hofker M.H. et al.Altered lipid metabolism in apolipoprotein E-deficient mice does not affect cholesterol balance across the liver.Hepatology. 1996; 24: 241-247Crossref PubMed Google Scholar Bile acid analysis was performed as previously described.38Mashige F. Imai K. Osuga T. A simple and sensitive assay of total serum bile acids.Clin Chim Acta. 1976; 70: 79-86Crossref PubMed Scopus (319) Google Scholar Cholesterol and phospholipid concentrations in the bile were determined as described.39Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification.Can J Biochem Physiol. 1959; 37: 911-917Crossref PubMed Scopus (43498) Google Scholar, 40Böttcher C. van Gent C.M. Pries C. A rapid and sensitive sub-micro phosphorus determination.Anal Chim Acta. 1961; 24: 203-204Crossref Scopus (855) Google Scholar, 41Gamble W. Vaughan M. Kruth H.S. et al.Procedure for determination of free and total cholesterol in micro- or nanogram amounts suitable for studies with cultured cells.J Lipid Res. 1978; 19: 1068-1070Abstract Full Text PDF PubMed Google Scholar Mice were housed individually for 48 hours, and total feces production was collected. Bile acids were extracted and analyzed as described.42Setchell K.D. Lawson A.M. Tanida N. et al.General methods for the analysis of metabolic profiles of bile acids and related compounds in feces.J Lipid Res. 1983; 24: 1085-1100Abstract Full Text PDF PubMed Google Scholar Microsomal proteins were isolated, and CYP7A1 protein was detected by immunoblotting using a specific antibody (see Supplementary Material online at www.gastrojournal.org). For RNA isolation and quantification, see Supplementary Material (see Supplementary Material online at www.gastrojournal.org). The mpSHPwtpGL3 construct was obtained by inserting a 0.4-kilobase fragment of the mouse SHP promoter (mSHPp wt) in the pGL3 vector (see Supplementary Material online at www.gastrojournal.org). The mSHPpmut pGL3 vector (mSHPp mut) was obtained by introducing a mutation in the Rev-erbα response element (RevRE) site using the RevREmut oligonucleotides (see Supplementary Material online at www.gastrojournal.org). The mSHPRevRE3xwt, mSHPRevRE3xmut, mE4BP4RevRE3xwt, and mE4BP4RevRE3xmut constructs were obtained by inserting 3 copies of the double-strand wild-type or mutated RevRE in the pTK-pGL3 plasmid. The human SHP promoter was a generous gift of Dr David Mangelsdorf.11Lu T.T. Makishima M. Repa J.J. et al.Molecular basis for feedback regulation of bile acid synthesis by nuclear receptors.Mol Cell. 2000; 6: 507-515Abstract Full Text Full Text PDF PubMed Scopus (1242) Google Scholar, 43Makishima M. Okamoto A.Y. Repa J.J. et al.Identification of a nuclear receptor for bile acids.Science. 1999; 284: 1362-1365Crossref PubMed Scopus (2209) Google Scholar The pCMX Rev-erbα expression vector was a gift of Dr Ron Evans, and the pSG5-hRev-erbα plasmid was as described.44Adelmant G. Begue A. Stehelin D. et al.A functional Rev-erbα responsive element located in the human Rev-erbα promoter mediates a repressing activity.Proc Natl Acad Sci U S A. 1996; 93: 3553-3558Crossref PubMed Scopus (100) Google Scholar, 45Issemann I. Green S. Activation of a member of the steroid hormone receptor superfamily by peroxisome proliferators.Nature. 1990; 347: 645-650Crossref PubMed Scopus (3074) Google Scholar Adenovirus construction details are given in the Supplementary Material (see Supplementary Material online at www.gastrojournal.org). Rev-erbα or green fluorescent protein (GFP) adenovirus was injected into male SV129 mice via the tail vein (1.108 plaque-forming units/mouse). Mice were killed 66 hours postinfection, and tissue collection and gene expression analysis were conducted as described in the previous paragraphs. RK-13 or HepG2 cells were transfected in Dulbecco's modified Eagle medium (DMEM) for 2 hours using RPR (120535B; Rhône-Poulenc Rorer, Annecy, France) or in medium using the Fugene reagent as previously described.31Raspe E. Duez H. Mansen A. et al.Identification of Rev-erbα as a physiological repressor of apoC-III gene transcription.J Lipid Res. 2002; 43: 2172-2179Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 46Claudel T. Sturm E. Duez H. et al.Bile acid-activated nuclear receptor FXR suppresses apolipoprotein A-I transcription via a negative FXR response element.J Clin Invest. 2002; 109: 961-971Crossref PubMed Scopus (290) Google Scholar A reporter/expression vector ratio of 1:5 was used. After 40 hours, cells were washed, and the luciferase activity was measured and normalized (see Supplementary Material online at www.gastrojournal.org). For details on electrophoretic mobility shift assays (EMSA), see Supplementary Material (see Supplementary Material online at www.gastrojournal.org). Perfused mouse livers were homogenized in extraction buffer, and nuclei were harvested by centrifugation and cross-linked with 1% formaldehyde. The reaction was stopped by addition of glycine at a final concentration of 200 mmol/L. After washing the nuclei, DNA was sheared by sonication and the supernatant precleared with a 50% protein A Sepharose slurry. Next, beads were removed, and immune complexes were allowed to form overnight with an anti-Rev-Erbα (Cell Signalling Technologies, Inc, Danvers, MA) or an anti-HA as negative control antibody (Santa Cruz Biotechnology, Inc, Santa Cruz, CA). The BMAL1, SHP, and E4BP4 or a nonspecific DNA region, as negative control, were amplified by qualitative polymerase chain reaction (QPCR) using specific primers (see Supplementary data online at www.gastrojournal.org). Statistical analyses were performed using the SPSS version 13.0 for Windows software (SPSS, Inc, Chicago, IL). Deficient vs wild-type or treated vs untreated groups were compared by the Student t test. When more than 2 groups were compared, analysis of variance (ANOVA) was used, and, when overall significance was attained, the Scheffe test was used to analyze for significant differences between the experimental groups. Whether Rev-erbα plays a role in bile acid metabolism was analyzed in vivo in Rev-erbα-deficient mice. Body weights were not different between age-matched chow-fed Rev-erbα-deficient and wild-type littermates (24.4 ± 2.4 vs 22.4 ± 0.5 g, respectively, P > .05). Rev-erbα-deficient mice displayed a significantly decreased bile flow (Rev-erbα−/−: 4.93 ± 0.75 vs Rev-erbα+/+: 6.33 ± 1.51 μL/min/100 g body weight, P < .05) and biliary excretion rate of bile acids (Rev-erbα−/−: 262 ± 78 vs Rev-erbα+/+: 526 ± 234 nmol/min/100 g body weight, P < .05) (Figure 1A and B). Biliary cholesterol and phospholipid contents, when normalized for bile acid content, did not significantly differ between the 2 genotypes (Figure 1C). Analysis of biliary bile acid composition revealed a 25% decrease in cholic acid concentrations in the Rev-erbα-deficient mice (Figure 1D). By contrast, no significant changes were found for the α, β, and ω-muricholic acids (Figure 1D), resulting in reduced cholate/ω-muricholate and cholate/β-muricholate ratios in Rev-erbα-deficient mice compared with their control wild-type littermates (cholate/ω-muricholate, Rev-erbα+/+: 16.93 vs Rev-erbα−/−: 10.66; cholate/β-muricholate, Rev-erbα+/+: 2.36 vs Rev-erbα−/−: 1.89). A significantly decreased fecal bile acid excretion was observed in Rev-erbα-deficient mice compared with wild-type littermates (Rev-erbα−/−: 1.65 ± 0.27 vs Rev-erbα+/+: 2.51 ± 0.52 μmol/day, P < .01) (Figure 1E) because of a significantly decreased excretion of cholic acid and its major metabolite deoxycholic acid (Figure 1F). Hepatic expression of proteins involved in bile acid transport, such as the Na+-taurocholate cotransporting polypeptide (NTCP) and the bile salt export pump (BSEP or ABCB11), was not altered by Rev-erbα deficiency (see Supplementary Figure S1 online at www.gastrojournal.org). Likewise, hepatic expression of genes involved in biliary excretion of cholesterol (ABCG5/ABCG8) and phospholipids (MDR-2 or ABCB4) was also not affected by Rev-erbα deficiency (see Supplementary Figure S1 online at www.gastrojournal.org). These data demonstrate impaired bile acid excretion into bile and feces in Rev-erbα-deficient mice, without alteration in transporters expression. Because, under steady-state conditions, fecal bile acid loss is a direct reflection of the hepatic bile acid synthesis rate, these results demonstrate that Rev-erbα deficiency is associated with impaired bile acid synthesis. CYP7A1 mRNA levels were measured in livers of chow-fed Rev-erbα-deficient mice and compared with wild-type mice (Figure 2A). Because Rev-erbα expression shows strong circadian variations, samples were collected in the afternoon, ie, at 4 pm when Rev-erbα expression is maximal.36Preitner N. Damiola F. Lopez-Molina L. et al.The orphan nuclear receptor REV-ERBZα controls circadian transcription within the positive limb of the mammalian circadian oscillator.Cell. 2002; 110: 251-260Abstract Full Text Full Text PDF PubMed Scopus (1718) Google Scholar Rev-erbα deficiency was associated with markedly decreased (−64%) hepatic CYP7A1 mRNA levels and hepatic microsomal CYP7A1 protein (Figure 2A). This decrease is coherent with the observed diminished biliary cholic acid concentration and fecal bile acid excretion. In contrast, CYP8B1 hepatic expression remained unchanged in Rev-erbα-deficient mice (Figure 2B). mRNA levels of CYP27A1, encoding the first enzyme of the acidic pathway of bile acid synthesis, were also not affected by Rev-erbα deficiency concordant with the absence of differences in muricholic acids (Figure 2C). Hepatic mRNA levels of Bmal1, a known Rev-erbα target gene,36Preitner N. Damiola F. Lopez-Molina L. et al.The orphan nuclear receptor REV-ERBZα controls circadian transcription within the positive limb of the mammalian circadian oscillator.Cell. 2002; 110: 251-260Abstract Full Text Full Text PDF PubMed Scopus (1718) Google Scholar were increased in Rev-erbα-deficient mice (see Supplementary Figure S1 online at www.gastrojournal.org). To assess whether Rev-erbα overexpression would induce an opposite regulation, wild-type mice were infected with a Rev-erbα adenovirus, and liver CYP7A1 mRNA levels were measured. In accordance with the above data, CYP7A1 mRNA levels were significantly increased in Rev-erbα-overexpressing mice compared with control GFP adenovirus-infected mice (Figure 2D and see Supplementary Figure S2 online at www.gastrojournal.org). By contrast, CYP8B1 and CYP27A1 hepatic gene expression were not affected by Rev-erbα overexpression (Figure 2E and F). Altogether, these data indicate that Rev-erbα positively regulates CYP7A1 expression in vivo. To delineate further the role of Rev-erbα in CYP7A1 regulation, Rev-erbα-deficient mice and wild-type littermates were treated for 5 days with 0.5% TCA. No changes in body weight or food intake were recorded (data not shown). In accordance with the previous results, CYP7A1 mRNA levels were significantly lower in Rev-erbα-deficient mice compared with controls. Furthermore and as previously described, CYP7A1 mRNA (Figure 3A) and protein (Figure 3B) levels markedly decreased in wild-type mice upon TCA treatment, reflecting the well-characterized retroinhibition of bile acid synthesis. Strikingly, TCA-induced decrease of CYP7A1 mRNA levels was highly enhanced in Rev-erbα-deficient mice, ie, an almost 10-fold repression, and CYP7A1 protein was barely detectable in microsomes of TCA-treated Rev-erbα-deficient mice (Figure 3B). Together, these results suggest that Rev-erbα may attenuate the bile acid-mediated repression of CYP7A1 expression and that this counteracting mechanism is abolished in the Rev-erbα-deficient mice, leading to an exacerbated repression of CYP7A1 by its downstream products. Because Rev-erbα is a core component of the biologic clock whose expression cycles at a high amplitude, we next examined whether Rev-erbα deficiency affects the circadian rhythms of CYP7A1 expression. As previously shown, CYP7A1 gene expression displayed a diurnal oscillation in wild-type mice, with a zenith in the early dark phase (Figure 4A, ZT13) and a minimum in the late dark phase (Figure 4A). CYP7A1 mRNA levels also cycled in Rev-erbα-deficient mice, and no phase shift or difference in amplitude was observed between the 2 genotypes. Moreover, the expression of CYP7A1 was lower at all time points in Rev-erbα-deficient mice except at ZT19, which corresponds to Rev-erbα lowest expression (Figure 4A and B). Circadian expression of Bmal1 also shows an altered pattern in Rev-erbα-deficient mice as previously reported36Preitner N. Damiola F. Lopez-Molina L. et al.The orphan nuclear receptor REV-ERBZα controls circadian transcription within the positive limb of the mammalian circadian oscillator.Cell. 2002; 110: 251-260Abstract Full Text Full Text PDF PubMed Scopus (1718) Google Scholar (see Supplementary Figure S3 online at www.gastrojournal.org). CYP7A1 gene promoter is under transcriptional control by transcription factors belonging to the master clock system as well as by several nuclear receptors. We therefore examined expression of the clock output genes DBP (a positive modulator of CYP7A1 transcription), Dec2, and E4BP4 (2 transcriptional repressors) as well as the nuclear receptors HNF4α and LXRα (positive regulators of CYP7A1) and peroxisomal proliferator-activated receptor (PPAR) α and SHP (negative regulators of CYP7A1) in livers from Rev-erbα-deficient mice at 4 pm. Rev-erbα deficiency did not affect PPARα, LXRα, or HNF4α or DBP or Dec2 mRNA levels (see Supplementary Figure S4 online at www.gastrojournal.org) but significantly increased liver E4BP4 and SHP mRNA levels (Figure 4C and D). Moreover, the expression of hepatic SHP and E4BP4 revealed circadian oscillations in wild-type mice, with both peaking in the opposite phase compared with Rev-erbα expression (Figure 4E and F). The oscillation in E4BP4 expression was abolished in Rev-erbα-deficient mice, whereas the phase of liver SHP mRNA accumulation was delayed by approximately 6 hours in Rev-erbα-deficient mice. These data confirm that both SHP and E4BP4 are under direct control of Rev-erbα. When the mouse and human SHP as well as the mouse E4BP4 promoters were analyzed for the presence of potential Rev-erbα binding sites resembling a hexameric core motif consisting of the consensu
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