RESUMEN
The nuclear receptor Farnesoid x receptor (FXR) is a critical regulator of multiple genes involved in bile acid homeostasis. The coactivators attracted to promoters of FXR target genes and epigenetic modifications that occur after ligand binding to FXR have not been completely defined, and it is unknown whether these processes are disrupted during cholestasis. Using a microarray, we identified decreased expression of mixed lineage leukemia 3 (MLL3), a histone H3 lysine 4 (H3K4) lysine methyl transferase at 1 and 3 days of post-common bile duct ligation (CBDL) in mice. Chromatin immunoprecipitation analysis (ChIP) analysis revealed that H3K4me3 of transporter promoters by MLL3 as part of activating signal cointegrator-2 -containing complex (ASCOM) is essential for activation of bile salt export pump (BSEP), multidrug resistance associated protein 2 (MRP2), and sodium taurocholate cotransporting polypeptide (NTCP) genes by FXR and glucocorticoid receptor (GR). Knockdown of nuclear receptor coactivator 6 (NCOA6) or MLL3/MLL4 mRNAs by small interfering RNA treatment led to a decrease in BSEP and NTCP mRNA levels in hepatoma cells. Human BSEP promoter transactivation by FXR/RXR was enhanced in a dose-dependent fashion by NCOA6 cDNA coexpression and decreased by AdsiNCOA6 infection in HepG2 cells. GST-pull down assays showed that domain 3 and 5 of NCOA6 (LXXLL motifs) interacted with FXR and that the interaction with domain 5 was enhanced by chenodeoxycholic acid. In vivo ChIP assays in HepG2 cells revealed ligand-dependent recruitment of ASCOM complex to FXR element in BSEP and GR element in NTCP promoters, respectively. ChIP analysis demonstrated significantly diminished recruitment of ASCOM complex components and H3K4me3 to Bsep and Mrp2 promoter FXR elements in mouse livers after CBDL. Taken together, these data show that the "H3K4me3" epigenetic mark is essential to activation of BSEP, NTCP, and MRP2 genes by nuclear receptors and is downregulated in cholestasis.
Asunto(s)
Proteínas Portadoras/genética , Colestasis/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/metabolismo , Glicoproteínas de Membrana/genética , Coactivadores de Receptor Nuclear/metabolismo , Receptores Citoplasmáticos y Nucleares/fisiología , Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP , Transportadoras de Casetes de Unión a ATP/biosíntesis , Transportadoras de Casetes de Unión a ATP/genética , Animales , Conductos Biliares/fisiología , Células Cultivadas , Colestasis/genética , Regulación hacia Abajo , Glutatión Transferasa/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , Humanos , Inmunoprecipitación , Ligadura , Metilación , Ratones , Coactivadores de Receptor Nuclear/genética , Transportadores de Anión Orgánico Sodio-Dependiente/biosíntesis , Transportadores de Anión Orgánico Sodio-Dependiente/genética , Plásmidos/genética , ARN Interferente Pequeño/genética , Receptores Citoplasmáticos y Nucleares/genética , Simportadores/biosíntesis , Simportadores/genéticaRESUMEN
Bile acids are the major determinant and driving force for the generation of bile flow. Bile acid transport across the canalicular membrane is primarily an ATP-dependent process. The predominant transporter is the bile salt excretory pump (BSEP, ABCB11), a member of the adenosine triphosphate-binding cassette (ABC) family of transporters. Regulatory mechanisms that can coordinate the genes encoding bile acid transport proteins are critically important to avoid hepatocyte damage from intracellar accumulation of bile acids. Bile salts are natural ligands for several nuclear hormone receptors expressed in liver and intestine. Nuclear receptors are transcription factors that bind specific ligands such as bile acids and regulate gene expression according to the metabolic requirements of the cell. In cloning of the BSEP gene, we found a binding site in the promoter for the farnesoid X receptor (FXR), a nuclear receptor for bile acids. FXR activity requires heterodimerization with the 9-cis retinoid receptor (RXR alpha), and when bound by bile acids and retinoic acid, the complex effectively activates the transcription of BSEP. There is a growing body of evidence for the activation of nuclear hormone receptors through the remodeling of chromatin by histone modification involving acetylation, in concert with methylation of H3 and H4 histones. We have recently demonstrated a role for the coactivator-associated arginine methyltransferase 1 (CARM1), as a coactivator of the FXR/RXR receptor and regulator of FXR responsive genes such as BSEP. Chromatin immunoprecipitation showed that the bile acid-dependent activation of the human BSEP is associated with a simultaneous increase of FXR and CARM1 occupation of the BSEP promoter. The increased occupation of the BSEP locus by CARM1 also corresponds with the increased deposition of Arg-17 methylation and Lys-9 acetylation of histone H3 within the FXR DNA-binding element of BSEP. Our work on the role of nuclear receptors in regulation of bile acid homeostasis has led to an increased understanding of the pathogenesis of the disorder, progressive familial intrahepatic cholestasis, type 1 (PFIC1) or Byler disease. The gene mutated in PFIC1 is called FIC1 and codes for a type IV P-type ATPase whose function is unknown. Increased ileal apical sodium-dependent bile acid transporter messenger RNA (mRNA) expression was detected in 3 patients with PFIC1. Ileal FXR and short heterodimer partner (an inhibitory nuclear receptor) messenger RNA levels were reduced in the same 3 patients. In studies of cells after antisense-mediated knock-down of endogenous FIC1, the activity of the ileal apical bile acid transporter promoter was enhanced, whereas the activities of the human FXR and BSEP promoters were reduced. Nuclear but not cytoplasmic localization of FXR is markedly decreased in FIC1-negative cells, indicating that FIC1 is necessary for posttranslational modifications necessary for the nuclear translocation of FXR. This defect leads to enhanced ileal bile salt uptake and impaired canalicular bile salt secretion by BSEP. In PFIC1, an increased load of bile acids is retained in the liver leading to cholestasis and progressive liver injury.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Ácidos y Sales Biliares/genética , Proteínas de Unión al ADN/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptor alfa X Retinoide/metabolismo , Factores de Transcripción/metabolismo , Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP , Transportadoras de Casetes de Unión a ATP/genética , Acetilación , Adenosina Trifosfatasas/metabolismo , Animales , Ácidos y Sales Biliares/metabolismo , Colestasis Intrahepática/metabolismo , Inmunoprecipitación de Cromatina , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Histonas/metabolismo , Humanos , Metilación , Regiones Promotoras Genéticas , Proteína-Arginina N-Metiltransferasas/metabolismo , ARN Mensajero/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Receptor alfa X Retinoide/genética , Factores de Transcripción/genética , Transcripción Genética , Activación TranscripcionalRESUMEN
Organic anion transporting polypeptides (Oatp) mediate the transport of a wide variety of amphipathic organic substrates. Rat Oatp1b2 and human OATP1B3 are members of a liver-specific subfamily of Oatps/OATPs. We investigated whether prolactin (PRL) and growth hormone (GH) regulated Oatp1b2 and OATP1B3 gene expression via signal transducers and activators of transcription 5 (Stat5). Binding sites for Stat5 transcription factors were located in the promoters of Oatp1b2 and OATP1B3 at -209 to -201 (5'-TTCTGGGAA-3') and -170 to -162 (5'-TTCTGAGAA-3'), respectively. In primary hepatocytes from female and male rats treated with PRL or GH, Oatp1b2 mRNA measured by real-time polymerase chain reaction was significantly induced 2-fold. HepG2 cells were transiently transfected with expression vectors containing Oatp1b2 or OATP1B3 promoter fragments, cDNAs for Stat5a, and the receptors for PRL (PRLR(L)) or GH (GHR), and treated with PRL or GH. PRL and GH induction of Oatp1b2 and OATP1B3 promoter activity required cotransfection of Stat5a and PRLR(L) or GHR. Mutation of the Stat5 binding site in both promoters eliminated hormonal induction. In DNA binding assays, HepG2 cells transfected with cDNAs for Stat5a and PRLR(L) were treated with PRL, and nuclear extracts were probed with a (32)P-labeled oligomer corresponding to -177 to -157 of the OATP1B3 promoter. PRL enhanced the binding of Stat5a to the OATP1B3 promoter and DNA-protein binding was inhibited in competition assays by excess OATP1B3 and Stat5 consensus oligomers but not by mutant Stat5 oligomers. These findings indicate that PRL and GH can regulate Oatp1b2 and OATP1B3 gene expression via the Stat5 signal-transduction pathway.
Asunto(s)
Hormona del Crecimiento/farmacología , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Transportadores de Anión Orgánico/metabolismo , Prolactina/farmacología , Animales , Células Cultivadas , Relación Dosis-Respuesta a Droga , Femenino , Masculino , Transportadores de Anión Orgánico/biosíntesis , Transportadores de Anión Orgánico/genética , Transportadores de Anión Orgánico Sodio-Independiente/biosíntesis , Transportadores de Anión Orgánico Sodio-Independiente/genética , Embarazo , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-DawleyRESUMEN
Nuclear receptors (NRs) play pivotal roles in the regulation of genes contributing to hepatobiliary cholesterol and bile acid homeostasis. We have previously shown that transporters involved in bile formation are developmentally regulated and are poorly developed during the fetal stage, but their expression reached gradual maturity during the postnatal period. To define the molecular mechanisms underlying this regulation and the role that class II NRs and associated members [liver receptor homolog-1 (LRH-1) and short heterodimer partner (SHP)] play, we have analyzed the ontogeny of NR expression during liver development. Real-time PCR analysis of hepatic NR expression from fetal day 17 through adult revealed that steady-state mRNA levels for all NRs were very low during the embryonic period. However, mRNA levels peaked close to that of adult rats (>6 wk-old rats) by 4 wk of age for farnesoid X receptor (FXR), pregnane X receptor (PXR), liver X receptor-alpha (LXRalpha), peroxisome proliferator-activated receptor-alpha (PPARalpha), retinoid acid receptor-alpha (RARalpha), LRH-1, and SHP, whereas RXRalpha mRNA lagged behind. FXR, PXR, LXRalpha, RARalpha, and PPARalpha functional activity in liver nuclear extracts assayed by gel EMSA demonstrated that the activity attained adult levels by 4 wk of age, exhibiting a strict correlation with mRNA levels. Surprisingly, PPARalpha activity was delayed as seen by EMSA assay. Protein levels for NRs also corresponded to the mRNA and functional activity except for RXRalpha. RXRalpha protein levels were higher than message levels, suggesting increased protein stability. We conclude that expression of NRs during rat liver development is primarily regulated by transcriptional mechanisms, which in turn, control the regulation of bile acid and cholesterol metabolic pathways.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica/fisiología , Hígado/metabolismo , Receptores Citoplasmáticos y Nucleares/biosíntesis , Animales , Femenino , Hígado/embriología , Hígado/crecimiento & desarrollo , Masculino , ARN Mensajero/metabolismo , RatasRESUMEN
The regulation of the rabbit apical sodium-dependent bile acid transporter (ASBT) was studied both in vivo and in vitro. New Zealand White rabbits were fed 0.5% deoxycholic acid (DCA) or SC-435, a competitive ASBT inhibitor, for 1 wk. In DCA-fed rabbits, ASBT expression was repressed, associated with activated FXR, and evidenced by increased ileal short heterodimer partner (SHP) mRNA. Feeding SC-435 to the rabbits blocked bile acid absorption, decreased SHP mRNA, and increased ASBT expression. A 1.9-kb rabbit ASBT 5'-flanking region (promoter) was cloned, and a cis-acting element for alpha-fetoprotein transcription factor (FTF) was identified (-1166/-1158). The effects of transcriptional factors and different bile acids on the rabbit ASBT promoter were studied in Caco-2 cells. FTF stimulated the rabbit ASBT promoter activity fourfold but not after the FTF binding site was deleted from the promoter. Increasing the SHP protein notably inhibited FTF-dependent trans-activation of rabbit ASBT. Adding hydrophobic bile acids deoxycholic acid, chenodeoxycholic acid, and cholic acid, activating ligands for FXR, inhibited rabbit ASBT promoter activity in Caco-2 cells, but this inhibitory effect was abolished after the FTF binding site was deleted. Ursodeoxycholic acid and ursocholic acid, nonactivating ligands for FXR, did not repress ASBT promoter activity. Thus the rabbit ASBT promoter is negative-feedback regulated by bile acids via a functional FTF binding site. Only FXR-activating ligands can downregulate rabbit ASBT expression through the regulatory cascade FXR-SHP-FTF.
Asunto(s)
Proteínas Portadoras/biosíntesis , Proteínas Portadoras/genética , Proteínas de Unión al ADN/farmacología , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/genética , Factores de Transcripción/farmacología , Animales , Ácidos y Sales Biliares/farmacología , Células CACO-2 , Proteínas Portadoras/farmacología , Ácido Desoxicólico/farmacología , Regulación hacia Abajo , Humanos , Ligandos , Masculino , Glicoproteínas de Membrana/farmacología , Regiones Promotoras Genéticas , Conejos , Receptores Citoplasmáticos y Nucleares/fisiología , alfa-FetoproteínasRESUMEN
BACKGROUND: Obese Zucker rats (ZR) have been used as an experimental model for non-alcoholic fatty liver disease and are particularly susceptible to various types of liver injury. Bile secretory function has not been assessed in ZR. AIM: To study bile secretion and expression of the main hepatobiliary transporters in ZR. METHODS: Bile flow and biliary secretion of lipids and glutathione were determined in eight and 14 week old obese ZR and their lean controls. Protein mass and mRNA of the Na(+)/taurocholate cotransporting polypeptide (Ntcp), the bile salt export pump (Bsep), and the multidrug resistant associated protein 2 (Mrp2) were assessed by western and northern blot, respectively. The effects of administration of a tumour necrosis factor alpha inactivator (etanercept) and an insulin sensitiser (rosiglitazone) were assessed in obese ZR while leptin was given to non-obese rats to study its effect on Mrp2 expression. RESULTS: ZR exhibited increased body weight and hyperlipidaemia. Only 14 week old obese ZR has fatty liver. Decreased bile flow and biliary lipid and glutathione secretion as well as reduced hepatic transport of both taurocholate and bromosulphthalein were found in obese ZR. Hepatic Mrp2 protein mass was markedly reduced (-70%) in obese rats while Ntcp and Bsep protein levels were similar to lean rats. Downregulation of Mrp2 seems to involve both transcriptional and post-transcriptional mechanisms probably related to insulin and leptin resistance. CONCLUSIONS: Obese ZR exhibit an impaired bile secretory function with significant functional and molecular alterations consistent with mild cholestasis. A defective hepatobiliary transport capacity may be a contributory factor in rendering the obese ZR more susceptible to liver injury.
Asunto(s)
Canalículos Biliares/metabolismo , Bilis/metabolismo , Colestasis/metabolismo , Obesidad/metabolismo , Animales , Transporte Biológico Activo , Peso Corporal , Colestasis/etiología , Colestasis/patología , Regulación hacia Abajo , Hígado/patología , Masculino , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Obesidad/complicaciones , Obesidad/patología , Tamaño de los Órganos , ARN Mensajero/genética , Ratas , Ratas ZuckerRESUMEN
BACKGROUND & AIMS: The mechanisms by which mutations in the familial intrahepatic cholestasis-1 gene cause Byler's disease (progressive familial intrahepatic cholestasis type 1) are unknown. METHODS: Interactions among the apical sodium-dependent bile acid transporter, the farnesoid X receptor (FXR), and familial intrahepatic cholestasis-1 were studied in the ileum of children with progressive familial intrahepatic cholestasis type 1 and in Caco-2 cells. RESULTS: Increased ileal apical sodium-dependent bile acid transporter messenger RNA (mRNA) expression was detected in 3 patients with progressive familial intrahepatic cholestasis type 1. Paradoxically, ileal lipid-binding protein mRNA expression was repressed, suggesting a central defect in bile acid response. Ileal FXR and short heterodimer partner mRNA levels were reduced in the same 3 patients. In Caco-2 cells, antisense-mediated knock-down of endogenous familial intrahepatic cholestasis-1 led to up-regulation of apical sodium-dependent bile acid transporter and down-regulation of FXR, ileal lipid-binding protein, and short heterodimer partner mRNA. In familial intrahepatic cholestasis-1-negative Caco-2 cells, the activity of the human apical sodium-dependent bile acid transporter promoter was enhanced, whereas the human FXR and bile salt excretory pump promoters' activities were reduced. Overexpression of short heterodimer partner but not of the FXR abrogated the effect of familial intrahepatic cholestasis-1 antisense oligonucleotides. FXR cis-element binding and FXR protein were reduced primarily in nuclear but not cytoplasmic extracts from familial intrahepatic cholestasis-1-negative Caco-2 cells. CONCLUSIONS: Loss of familial intrahepatic cholestasis-1 leads to diminished nuclear translocation of the FXR, with the subsequent potential for pathologic alterations in intestinal and hepatic bile acid transporter expression. Marked hypercholanemia and cholestasis are predicted to develop, presumably because of both enhanced ileal uptake of bile salts via up-regulation of the apical sodium-dependent bile acid transporter and diminished canalicular secretion of bile salts secondary to down-regulation of the bile salt excretory pump.
Asunto(s)
Colestasis/genética , Colestasis/metabolismo , Proteínas de Unión al ADN/metabolismo , Transportadores de Anión Orgánico Sodio-Dependiente , Simportadores , Factores de Transcripción/metabolismo , Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP , Transportadoras de Casetes de Unión a ATP/genética , Adenosina Trifosfatasas/deficiencia , Adenosina Trifosfatasas/genética , Células CACO-2 , Proteínas Portadoras/genética , Núcleo Celular/metabolismo , Niño , Preescolar , Citoplasma/metabolismo , Proteínas de Unión al ADN/genética , Progresión de la Enfermedad , Regulación hacia Abajo , Humanos , Lactante , Mutación , Oligonucleótidos Antisentido/farmacología , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , Receptores Citoplasmáticos y Nucleares , Factores de Transcripción/genética , Regulación hacia ArribaRESUMEN
We investigated how cholesterol feeding regulates cholesterol 7alpha-hydroxylase (CYP7A1) via the nuclear receptors farnesoid X receptor (FXR) and liver X receptor alpha (LXRalpha) in New Zealand white rabbits. After 1 day of 2% cholesterol feeding, when the bile acid pool size had not expanded, mRNA levels of the FXR target genes short-heterodimer partner (SHP) and sterol 12alpha-hydroxylase (CYP8B) were unchanged, indicating that FXR activation remained constant. In contrast, the mRNA levels of the LXRalpha target genes ATP binding cassette transporter A1 (ABCA1) and cholesteryl ester transfer protein (CETP) increased 5-fold and 2.3-fold, respectively, associated with significant increases in hepatic concentrations of oxysterols. Activity and mRNA levels of CYP7A1 increased 2.4 times and 2.2 times, respectively. After 10 days of cholesterol feeding, the bile acid pool size increased nearly 2-fold. SHP mRNA levels increased 4.1-fold while CYP8B declined 64%. ABCA1 mRNA rose 8-fold and CETP mRNA remained elevated. Activity and mRNA of CYP7A1 decreased 60% and 90%, respectively. Feeding cholesterol for 1 day did not enlarge the ligand pool size or change FXR activation, while LXRalpha was activated highly secondary to increased hepatic oxysterols. As a result, CYP7A1 was up-regulated. After 10 days of cholesterol feeding, the bile acid (FXR ligand) pool size increased, which activated FXR and inhibited CYP7A1 despite continued activation of LXRalpha. Thus, in rabbits, when FXR and LXRalpha are activated simultaneously, the inhibitory effect of FXR overrides the stimulatory effect of LXRalpha to suppress CYP7A1 mRNA expression.
Asunto(s)
Colesterol 7-alfa-Hidroxilasa/metabolismo , Colesterol/administración & dosificación , Proteínas de Unión al ADN/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Factores de Transcripción/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Ácidos y Sales Biliares/metabolismo , Proteínas Portadoras/metabolismo , Colesterol/metabolismo , Proteínas de Transferencia de Ésteres de Colesterol , Regulación hacia Abajo , Glicoproteínas/metabolismo , Ligandos , Hígado/metabolismo , Receptores X del Hígado , Receptores Nucleares Huérfanos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Conejos , Receptores de Esteroides/metabolismo , Esteroide 12-alfa-Hidroxilasa/metabolismo , Factores de TiempoRESUMEN
Intestinal reclamation of bile salts is mediated in large part by the apical sodium-dependent bile acid transporter (ASBT). The bile acid responsiveness of ASBT is controversial. Bile acid feeding in mice results in decreased expression of ASBT protein and mRNA. Mouse but not rat ASBT promoter activity was repressed in Caco-2, but not IEC-6, cells by chenodeoxycholic acid. A potential liver receptor homologue-1 (LRH-1) cis-acting element was identified in the bile acid-responsive region of the mouse but not rat promoter. The mouse, but not rat, promoter was activated by LRH-1, and this correlated with nuclear protein binding to the mouse but not rat LRH-1 element. The short heterodimer partner diminished the activity of the mouse promoter and could partially offset its activation by LRH-1. Interconversion of the potential LRH-1 cis-elements between the mouse and rat ASBT promoters was associated with an interconversion of LRH-1 and bile acid responsiveness. LRH-1 protein was found in Caco-2 cells and mouse ileum, but not IEC-6 cells or rat ileum. Bile acid response was mediated by the farnesoid X receptor, as shown by the fact that overexpression of a dominant-negative farnesoid X-receptor eliminated the bile acid mediated down-regulation of ASBT. In addition, ASBT expression in farnesoid X receptor null mice was unresponsive to bile acid feeding. In summary cell line- and species-specific negative feedback regulation of ASBT by bile acids is mediated by farnesoid X receptor via small heterodimer partner-dependent repression of LRH-1 activation of the ASBT promoter.
Asunto(s)
Proteínas Portadoras/metabolismo , Ácido Quenodesoxicólico/administración & dosificación , Transportadores de Anión Orgánico Sodio-Dependiente , Receptores Citoplasmáticos y Nucleares/fisiología , Simportadores , Animales , Secuencia de Bases , Proteínas Portadoras/química , Proteínas Portadoras/genética , Línea Celular , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Ratas , Receptores Citoplasmáticos y Nucleares/genética , Homología de Secuencia de Ácido NucleicoRESUMEN
We investigated the role of the orphan nuclear receptor farnesoid X receptor (FXR) in the regulation of cholesterol 7alpha-hydroxylase (CYP7A1), using an in vivo rabbit model, in which the bile acid pool, which includes high affinity ligands for FXR, was eliminated. After 7 days of bile drainage, the enterohepatic bile acid pool, in both New Zealand White and Watanabe heritable hyperlipidemic rabbits, was depleted. CYP7A1 activity and mRNA levels increased while FXR was deactivated as indicated by reduced FXR protein and changes in the expression of target genes that served as surrogate markers of FXR activation in the liver and ileum, respectively. Hepatic bile salt export pump mRNA levels and ileal bile acid-binding protein decreased while sterol 12alpha-hydroxylase and sodium/taurocholate cotransporting polypeptide mRNA levels increased in the liver. In addition, hepatic FXR mRNA levels decreased significantly. The data, taken together, indicate that FXR was deactivated when the bile acid pool was depleted such that CYP7A1 was upregulated. Further, lack of the high affinity ligand supply was associated with downregulation of hepatic FXR mRNA levels.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Ácidos y Sales Biliares/metabolismo , Proteínas Portadoras/metabolismo , Colesterol 7-alfa-Hidroxilasa/metabolismo , Proteínas de Unión al ADN/metabolismo , Hidroxiesteroide Deshidrogenasas , Glicoproteínas de Membrana , Proteínas de Transporte de Membrana , Factores de Transcripción/metabolismo , Animales , Sistema Enzimático del Citocromo P-450/metabolismo , Drenaje/métodos , Masculino , Transportadores de Anión Orgánico Sodio-Dependiente , ARN Mensajero/metabolismo , Conejos , Receptores Citoplasmáticos y Nucleares , Esteroide 12-alfa-Hidroxilasa , Esteroide Hidroxilasas/metabolismo , Simportadores , Regulación hacia ArribaRESUMEN
To study the effect of cholecystectomy on the regulation of classic and alternative bile acid syntheses, gallbladder-intact (n = 20) and cholecystectomized (n = 20) New Zealand White rabbits were fed either chow or chow with 2% cholesterol (3 g/day). After 10 days, bile fistulas were constructed in half of each rabbit group to recover and measure the bile acid pool and biliary bile acid flux. After cholesterol feeding, the bile acid pool size increased from 268 +/- 55 to 444 +/- 77 mg (P < 0.01) with a 2-fold rise in the biliary bile acid flux in intact rabbits but did not expand the bile acid pool (270 +/- 77 vs. 276 +/- 62 mg), nor did the biliary bile acid flux increase in cholecystectomized rabbits. Ileal apical sodium-dependent bile acid transporter protein increased 46% from 93 +/- 6 to 136 +/- 23 units/mg (P < 0.01) in the intact rabbits but did not change in cholecystectomized rabbits (104 +/- 14 vs. 99 +/- 19 units/mg) after cholesterol feeding. Cholesterol 7alpha-hydroxylase activity was inhibited 59% (P < 0.001) while cholesterol 27-hydroxylase activity rose 83% (P < 0.05) after cholesterol feeding in the intact rabbits but neither enzyme activity changed significantly in cholesterol-fed cholecystectomized rabbits. Fecal bile acid outputs reflecting bile acid synthesis increased significantly in the intact but not in the cholecystectomized rabbits fed cholesterol. Removal of the gallbladder prevented expansion of the bile acid pool after cholesterol feeding as seen in intact rabbits because ileal bile acid transport did not increase. As a result, cholesterol 7alpha-hydroxylase was not inhibited.
Asunto(s)
Ácidos y Sales Biliares/biosíntesis , Colecistectomía , Colesterol 7-alfa-Hidroxilasa/antagonistas & inhibidores , Colesterol en la Dieta/administración & dosificación , Vesícula Biliar/fisiología , Hidroxiesteroide Deshidrogenasas , Glicoproteínas de Membrana , Proteínas de Transporte de Membrana , Animales , Ácidos y Sales Biliares/metabolismo , Western Blotting , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Colestanotriol 26-Monooxigenasa , Colesterol/sangre , Colesterol/metabolismo , Colesterol 7-alfa-Hidroxilasa/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Heces/química , Hidroximetilglutaril-CoA Reductasas/metabolismo , Íleon/metabolismo , Hígado/metabolismo , Masculino , Transportadores de Anión Orgánico Sodio-Dependiente , ARN Mensajero/análisis , Conejos , Esteroide Hidroxilasas/metabolismo , SimportadoresRESUMEN
The bile salt excretory pump (BSEP, ABCb11) is critical for ATP-dependent transport of bile acids across the hepatocyte canalicular membrane and for generation of bile acid-dependent bile secretion. Recent studies have demonstrated that the expression of this transporter is sensitive to the flux of bile acids through the hepatocyte, possibly at the level of transcription of the BSEP gene. To determine the mechanisms underlying the regulation of BSEP by bile acids, the promoter of the BSEP gene was cloned. The sequence of the promoter contained an inverted repeat (IR)-1 element (5'-GGGACA T TGATCCT-3') at base pairs -63/-50 consisting of two nuclear receptor half-sites organized as an inverted repeat and separated by a single nucleotide. This IR-1 element has been shown in several recent studies to serve as a binding site for the farnesoid X receptor (FXR), a nuclear receptor for bile acids. FXR activity requires heterodimerization with RXR alpha, and when bound by bile acids, the complex effectively regulates the transcription of several genes involved in bile acid homeostasis. Gel mobility shift assays demonstrated specific binding of FXR/RXR alpha heterodimers to the IR-1 element in the BSEP promoter. In HepG2 cells, co-transfection of FXR and RXR alpha is required to attain full transactivation of the BSEP promoter by bile acids. Two FXR transactivation-deficient mutants (an AF-2 deletion and a W469A point mutant) failed to transactivate, indicating that the effect of bile acids is FXR-dependent. Further, mutational analysis confirms that the FXR/RXR alpha heterodimer activates transcription through the IR-1 site in the human BSEP promoter. These results demonstrate a mechanism by which bile acids transcriptionally regulate the activity of the bile salt excretory pump, a critical component involved in the enterohepatic circulation of bile acids.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Proteínas de Unión al ADN/metabolismo , Regiones Promotoras Genéticas , Factores de Transcripción/metabolismo , Activación Transcripcional , Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP , Transportadoras de Casetes de Unión a ATP/metabolismo , Alitretinoína , Sustitución de Aminoácidos , Secuencia de Bases , Ácidos y Sales Biliares/metabolismo , Sitios de Unión , Carcinoma Hepatocelular , Cartilla de ADN , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica/efectos de los fármacos , Genes Reporteros , Hepatocitos/metabolismo , Humanos , Neoplasias Hepáticas , Datos de Secuencia Molecular , Mutagénesis , Mutagénesis Sitio-Dirigida , Mutación Puntual , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Ácido Retinoico/genética , Receptores de Ácido Retinoico/metabolismo , Proteínas Recombinantes/metabolismo , Receptores X Retinoide , Eliminación de Secuencia , TATA Box , Factores de Transcripción/genética , Transcripción Genética , Activación Transcripcional/efectos de los fármacos , Transfección , Tretinoina/farmacología , Células Tumorales CultivadasAsunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Colestasis Intrahepática/genética , Ratones Noqueados/genética , Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP , Animales , Bilis/metabolismo , Colestasis Intrahepática/metabolismo , Modelos Animales de Enfermedad , Metabolismo de los Lípidos , RatonesRESUMEN
Maturity-onset diabetes of the young type 3 (MODY3) is caused by haploinsufficiency of hepatocyte nuclear factor-1alpha (encoded by TCF1). Tcf1-/- mice have type 2 diabetes, dwarfism, renal Fanconi syndrome, hepatic dysfunction and hypercholestrolemia. Here we explore the molecular basis for the hypercholesterolemia using oligonucleotide microchip expression analysis. We demonstrate that Tcf1-/- mice have a defect in bile acid transport, increased bile acid and liver cholesterol synthesis, and impaired HDL metabolism. Tcf1-/- liver has decreased expression of the basolateral membrane bile acid transporters Slc10a1, Slc21a3 and Slc21a5, leading to impaired portal bile acid uptake and elevated plasma bile acid concentrations. In intestine and kidneys, Tcf1-/- mice lack expression of the ileal bile acid transporter (Slc10a2), resulting in increased fecal and urinary bile acid excretion. The Tcf1 protein (also known as HNF-1alpha) also regulates transcription of the gene (Nr1h4) encoding the farnesoid X receptor-1 (Fxr-1), thereby leading to reduced expression of small heterodimer partner-1 (Shp-1) and repression of Cyp7a1, the rate-limiting enzyme in the classic bile acid biosynthesis pathway. In addition, hepatocyte bile acid storage protein is absent from Tcf1-/- mice. Increased plasma cholesterol of Tcf1-/- mice resides predominantly in large, buoyant, high-density lipoprotein (HDL) particles. This is most likely due to reduced activity of the HDL-catabolic enzyme hepatic lipase (Lipc) and increased expression of HDL-cholesterol esterifying enzyme lecithin:cholesterol acyl transferase (Lcat). Our studies demonstrate that Tcf1, in addition to being an important regulator of insulin secretion, is an essential transcriptional regulator of bile acid and HDL-cholesterol metabolism.
Asunto(s)
Ácidos y Sales Biliares/metabolismo , Colesterol/sangre , Proteínas de Unión al ADN/fisiología , Proteínas Nucleares/fisiología , Animales , Secuencia de Bases , Ácidos y Sales Biliares/biosíntesis , Cartilla de ADN , Factor 1 Inducible por Hipoxia , Subunidad alfa del Factor 1 Inducible por Hipoxia , Íleon/metabolismo , Riñón/metabolismo , Ratones , Ratones Noqueados , Factores de Transcripción/genética , Factores de Transcripción/fisiologíaRESUMEN
In vivo tumor targetting with radiolabelled monoclonal antibodies is a promising approach for the diagnosis and therapy of tumors. A specific monoclonal antibody (mAb), DLAB was generated to the Dalton's lymphoma associated antigen (DLAA) from Haemophilus paragallinarum-induced spontaneous fusion. In order to study the tumor localisation and biodistribution properties of the monoclonal antibody, scintigraphic studies were performed using the radiolabelled DLAB. 131-labelled DLAB was administered intravenously into Swiss mice bearing Dalton's lymphoma and external scintiscanning was performed at different time intervals. Clear tumor images were obtained which revealed selective and specific uptake of radiolabel and the results were compared with biodistribution data. The radioiodinated monoclonal antibody showed fast tumor uptake which increased significantly to 14.6% injected dose (ID)/g at 12 hr post-injection. Enhanced blood clearance of radioactivity resulted in higher tumor/blood ratio of 5.96 at 48 hr. 131I-labelled DLAB resulted in selective and enhanced uptake of the radioactivity by the tumor compared to the non-specific antibody and the results suggest the potential use of spontaneous fusion for producing specific monoclonal antibodies for tumor detection and therapy.
Asunto(s)
Anticuerpos Monoclonales , Anticuerpos Antineoplásicos , Antígenos de Neoplasias/inmunología , Radioisótopos de Yodo , Linfoma/diagnóstico por imagen , Animales , Anticuerpos Monoclonales/farmacocinética , Modelos Animales de Enfermedad , Radioisótopos de Yodo/farmacocinética , Linfoma/inmunología , Ratones , Ratones Endogámicos DBA , Radioinmunodetección/métodos , Sensibilidad y EspecificidadRESUMEN
AIMS AND BACKGROUND: Radiolabeled antibodies generated against tumor-associated antigens are used for immunoscintigraphy to detect tumors and tumor metastases. Although successful tumor imaging has been achieved using trace-labeled murine monoclonal antibodies, such antibodies often lead to the development of human anti-murine antibodies (HAMA), which limit their subsequent administration for tumor imaging and therapy. It has been reported recently that chicken polyclonal antibodies have high affinity and specificity for the antigen against which they are raised and do not have any immunological cross-reactivity with HAMA. METHODS: The present study deals with immunoscintigraphy of Dalton's lymphoma, an experimental tumor model using chicken antibodies generated against Dalton's lymphoma-associated antigen (DLAA) and labeled with technetium-99m ((99m)Tc). RESULTS: Scintigrams showed specific uptake of the radiolabel resulting in clear tumor images. The radioactivity uptake of the chicken anti-DLAA antibody was about twofold higher than that of the non-specific chicken antibody. CONCLUSIONS: The results demonstrate the potential of chicken antibody for in vivo radioimmunodetection and localization of tumors.
Asunto(s)
Anticuerpos , Pollos/inmunología , Linfoma/diagnóstico por imagen , Radioinmunodetección/métodos , Tecnecio , Animales , Reacciones Antígeno-Anticuerpo , Ascitis/diagnóstico por imagen , Modelos Animales de Enfermedad , Inmunoglobulina G , Ratones , Factores de Tiempo , Distribución TisularRESUMEN
Extracellular adenosine triphosphate (ATP) may regulate hepatocyte and cholangiocyte functions, and under some conditions it may have deleterious effects on bile secretion and cause cholestasis. The canalicular membrane enzyme Ca2+/Mg2+-ecto-ATPase (ecto-ATPase) hydrolyzes ATP/adenosine diphosphate (ATP/ADP) and regulates hepatic extracellular ATP concentration. Changes in liver ecto-ATPase in estrogen-induced cholestasis were examined in male rats receiving 17alpha-ethinylestradiol (E groups) for 1, 3, or 5 days (5 mg/kg/day, sc) and compared with changes in rats subjected to obstructive cholestasis (O groups) for 1, 3, or 8 days. Activity of ecto-ATPase, protein mass in canalicular membranes and bile (estimated by Western blotting), steady state mRNA levels (by Northern blotting), and cellular and acinar distributions of the enzyme (histochemistry and immunocytochemistry) were assessed in these groups. Activity of ecto-ATPase, protein mass in isolated canalicular membranes, and enzyme mRNA levels were significantly increased in E group rats as compared with controls. In contrast, these parameters were markedly decreased in O group rats, and the enzyme protein was undetectable in bile. The ecto-ATPase histochemical reaction was markedly increased in the canalicular membrane of E group rats, extending from acinar zone 2 to zone 1, whereas it decreased in the O group. The ecto-ATPase immunocytochemical reaction was present in the canalicular membrane and pericanalicular vesicles in control and E group hepatocytes, but it decreased in obstructive cholestasis and was localized only to the canalicular membrane. Thus, significant changes in liver ecto-ATPase were apparent in 17alpha-ethinylestradiol-induced cholestasis that were opposite to those observed in obstructive cholestasis. Assuming that the alterations observed in obstructive cholestasis are the result of the cholestatic phenomenon, we conclude that changes in ecto-ATPase in 17alpha-ethinylestradiol-treated rats might be either primary events or part of an adaptive response in 17alpha-ethinylestradiol-induced cholestasis.
Asunto(s)
Adenosina Trifosfatasas/biosíntesis , Bilis/enzimología , Colestasis Extrahepática/inducido químicamente , Colestasis Extrahepática/enzimología , Etinilestradiol/toxicidad , Hígado/enzimología , Animales , Colestasis Extrahepática/patología , Inmunohistoquímica , Hígado/patología , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
Spontaneous cell fusion induced by the bacterium Haemophilus paragallinarum has been recently reported as an alternative technique to generate hybridomas producing monoclonal antibody (mAb). In order to investigate the advantages of this technique to produce anti-tumor monoclonal antibodies we performed comparative experiments between H. paragallinarum induced spontaneous cell fusion and polyethylene glycol (PEG) mediated fusion. Hybridomas producing monoclonal antibodies to an experimental murine lymphoma antigen, the Dalton's lymphoma associated antigen (DLAA) were generated and their sensitivity and specificity were ascertained. The spontaneous fusion yielded more number of stable and specific hybridomas than PEG mediated fusion. The results suggest the advantage of H. paragalinarum induced cell fusion for the simplified production of specific antitumor monoclonal antibodies.
Asunto(s)
Anticuerpos Antineoplásicos/biosíntesis , Antígenos de Neoplasias/biosíntesis , Haemophilus , Linfoma/inmunología , Animales , Anticuerpos Monoclonales/biosíntesis , Fusión Celular , Ensayo de Inmunoadsorción Enzimática , Antígenos de Histocompatibilidad Clase I/biosíntesis , Inmunoglobulina G/biosíntesis , Inmunoglobulina M/biosíntesis , Ratones , Ratones Endogámicos DBA , Sensibilidad y EspecificidadRESUMEN
Na+/taurocholate (Na+/TC) cotransport in hepatocytes is mediated primarily by Na+/TC cotransporting polypeptide (Ntcp), and cyclic adenosine monophosphate (cAMP) stimulates Na+/TC cotransport by inducing translocation of Ntcp to the plasma membrane. The aim of the present study was to determine if Ntcp is a phosphoprotein and if cAMP alters Ntcp phosphorylation. Freshly prepared hepatocytes from rat livers were incubated with carrier-free 32PO4 for 2 hours, followed by incubation with 10 micromol/L 8-chlorophenylthio adenosin 3':5'-cyclic monophosphate (CPT-cAMP) for 15 minutes. Subcellular fractions isolated from 32P-labeled hepatocytes were subjected to immunoprecipitation using Ntcp antibody, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography to determine if Ntcp is phosphorylated. Ntcp immunoprecipitated from plasma membranes isolated from nonlabeled hepatocytes was subjected to immunoblot analysis using anti-phosphoserine, anti-phosphothreonine, or anti-phosphotyrosine antibody to determine whether Ntcp is a serine, threonine, or tyrosine phosphoprotein. Hepatocytes were loaded with bis-(2-amino-5-methylphenoxy)-ethane-N,N,N',N'-tetraacetic acid (MAPTA), a Ca2+ buffering agent, and the effect of CPT-cAMP on TC uptake, cytosolic [Ca2+], and ntcp phosphorylation and translocation was determined. In addition, the effect of cAMP on protein phosphatases 1 and 2A (PP1/2A) was determined in homogenates and plasma membranes obtained from CPT-cAMP-treated hepatocytes. Phosphorylation study showed that phosphorylated Ntcp is detectable in plasma membranes, and cAMP treatment resulted in dephosphorylation of Ntcp. Immunoblot analysis with phosphoamino antibodies revealed that Ntcp is a serine/threonine, and not a tyrosine, phosphoprotein, and cAMP inhibited both serine and threonine phosphorylation. In MAPTA-loaded hepatocytes, CPT-cAMP failed to stimulate TC uptake, failed to increase cytosolic [Ca2+], and failed to induce translocation and dephosphorylation of Ntcp. cAMP did not alter the activity of PP1/2A in either homogenates or in plasma membranes. Taken together, these results suggest that Ntcp is a serine/threonine phosphoprotein and is dephosphorylated by cAMP treatment. Activation of PP1/2A is not involved in cAMP-mediated dephosphorylation of Ntcp. Both translocation and dephosphorylation of Ntcp may be involved in the regulation of hepatic Na+/TC cotransport.
Asunto(s)
Proteínas Portadoras/metabolismo , AMP Cíclico/metabolismo , Transportadores de Anión Orgánico Sodio-Dependiente , Fosfoproteínas/metabolismo , Serina/metabolismo , Simportadores , Treonina/metabolismo , Aminoácidos/análisis , Animales , Proteínas Portadoras/química , AMP Cíclico/farmacología , Hígado/citología , Hígado/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación/efectos de los fármacos , Ratas , Fracciones Subcelulares/metabolismo , Distribución TisularRESUMEN
The rat ileal apical Na+-dependent bile acid transporter (ASBT) and the liver Na+-taurocholate cotransporting polypeptide (Ntcp) are members of a new family of anion transporters. These transport proteins share limited sequence homology and almost identical predicted secondary structures but are localized to the apical surface of ileal enterocytes and the sinusoidal surface of hepatocytes, respectively. Stably transfected Madin-Darby canine kidney (MDCK) cells appropriately localized wild-type ASBT and Ntcp apically and basolaterally as assessed by functional activity and immunocytochemical localization studies. Truncated and chimeric transporters were used to determine the functional importance of the cytoplasmic tail in bile acid transport activity and membrane localization. Two cDNAs were created encoding a truncated transporter in which the 56-amino-acid COOH-terminal tail of Ntcp was removed or substituted with an eight-amino-acid epitope FLAG. For both mutants there was some loss of fidelity in basolateral sorting in that approximately 75% of each protein was delivered to the basolateral surface compared with approximately 90% of the wild-type Ntcp protein. In contrast, deletion of the cytoplasmic tail of ASBT led to complete loss of transport activity and sorting to the apical membrane. An Ntcp chimera in which the 56-amino-acid COOH-terminal tail of Ntcp was replaced with the 40-amino-acid cytoplasmic tail of ASBT was largely redirected (82.4 +/- 3.9%) to the apical domain of stably transfected MDCK cells, based on polarity of bile acid transport activity and localization by confocal immunofluorescence microscopy. These results indicate that a predominant signal for sorting of the Ntcp protein to the basolateral domain is located in a region outside of the cytoplasmic tail. These studies have further shown that a novel apical sorting signal is localized to the cytoplasmic tail of ASBT and that it is transferable and capable of redirecting a protein normally sorted to the basolateral surface to the apical domain of MDCK cells.