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1.
Hepatology ; 75(2): 252-265, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34387888

RESUMO

BACKGROUND AND AIMS: Parenteral nutrition (PN)-associated cholestasis (PNAC) complicates the care of patients with intestinal failure. In PNAC, phytosterol containing PN synergizes with intestinal injury and IL-1ß derived from activated hepatic macrophages to suppress hepatocyte farnesoid X receptor (FXR) signaling and promote PNAC. We hypothesized that pharmacological activation of FXR would prevent PNAC in a mouse model. APPROACH AND RESULTS: To induce PNAC, male C57BL/6 mice were subjected to intestinal injury (2% dextran sulfate sodium [DSS] for 4 days) followed by central venous catheterization and 14-day infusion of PN with or without the FXR agonist GW4064. Following sacrifice, hepatocellular injury, inflammation, and biliary and sterol transporter expression were determined. GW4064 (30 mg/kg/day) added to PN on days 4-14 prevented hepatic injury and cholestasis; reversed the suppressed mRNA expression of nuclear receptor subfamily 1, group H, member 4 (Nr1h4)/FXR, ATP-binding cassette subfamily B member 11 (Abcb11)/bile salt export pump, ATP-binding cassette subfamily C member 2 (Abcc2), ATP binding cassette subfamily B member 4(Abcb4), and ATP-binding cassette subfamily G members 5/8(Abcg5/8); and normalized serum bile acids. Chromatin immunoprecipitation of liver showed that GW4064 increased FXR binding to the Abcb11 promoter. Furthermore, GW4064 prevented DSS-PN-induced hepatic macrophage accumulation, hepatic expression of genes associated with macrophage recruitment and activation (ll-1b, C-C motif chemokine receptor 2, integrin subunit alpha M, lymphocyte antigen 6 complex locus C), and hepatic macrophage cytokine transcription in response to lipopolysaccharide in vitro. In primary mouse hepatocytes, GW4064 activated transcription of FXR canonical targets, irrespective of IL-1ß exposure. Intestinal inflammation and ileal mRNAs (Nr1h4, Fgf15, and organic solute transporter alpha) were not different among groups, supporting a liver-specific effect of GW4064 in this model. CONCLUSIONS: GW4064 prevents PNAC in mice through restoration of hepatic FXR signaling, resulting in increased expression of canalicular bile and of sterol and phospholipid transporters and suppression of macrophage recruitment and activation. These data support augmenting FXR activity as a therapeutic strategy to alleviate or prevent PNAC.


Assuntos
Colestase/prevenção & controle , Expressão Gênica/efeitos dos fármacos , Isoxazóis/farmacologia , Nutrição Parenteral/efeitos adversos , Receptores Citoplasmáticos e Nucleares/agonistas , Receptores Citoplasmáticos e Nucleares/genética , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/genética , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/metabolismo , Membro 5 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Membro 8 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Animais , Ácidos e Sais Biliares/sangue , Colestase/etiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Hepatócitos/metabolismo , Interleucina-1beta/farmacologia , Enteropatias/induzido quimicamente , Enteropatias/terapia , Isoxazóis/uso terapêutico , Lipoproteínas/genética , Hepatopatias/etiologia , Hepatopatias/patologia , Hepatopatias/prevenção & controle , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteína 2 Associada à Farmacorresistência Múltipla/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , RNA Mensageiro/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Transdução de Sinais/efeitos dos fármacos
2.
J Biol Chem ; 297(6): 101400, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34774795

RESUMO

ATP-binding cassette, subfamily B member 11 (ABCB11) is an efflux transporter for bile acids on the liver canalicular membrane. The expression of this transporter is reduced in cholestasis; however, the mechanisms contributing to this reduction are unclear. In this study, we sought to determine whether miR-199a-5p contributes to the depletion of ABCB11/Abcb11 in cholestasis in mice. In a microRNA (miRNA) screen of mouse liver after common bile duct ligation (CBDL), we found that miR-199a-5p was significantly upregulated by approximately fourfold. In silico analysis predicted that miR-199a-5p would target the 3'-untranslated region (3'-UTR) of ABCB11/Abcb11 mRNA. The expression of ABCB11-3'-UTR luciferase construct in Huh-7 cells was markedly inhibited by cotransfection of a miRNA-199a-5p mimic, which was reversed by an miRNA-199a-5p mimic inhibitor. We also show treatment of mice after CBDL with the potent nuclear receptor FXR agonist obeticholic acid (OCA) significantly increased Abcb11 mRNA and protein and decreased miR-199a-5p expression. Computational mapping revealed a well-conserved FXR-binding site (FXRE) in the promoter of the gene encoding miR-199a-5, termed miR199a-2. Electromobility shift, chromatin immunoprecipitation, and miR199a-2 promoter-luciferase assays confirmed that this binding site was functional. Finally, CBDL in mice led to depletion of nuclear repressor NcoR1 binding at the miR199a-2 promoter, which facilitates transcription of miR199a-2. In CBDL mice treated with OCA, NcoR1 recruitment to the miR199a-2 FXRE was maintained at levels found in sham-operated mice. In conclusion, we demonstrate that miR-199a-5p is involved in regulating ABCB11/Abcb11 expression, is aberrantly upregulated in obstructive cholestasis, and is downregulated by the FXR agonist OCA.


Assuntos
Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/biossíntese , Colestase/metabolismo , Regulação da Expressão Gênica , MicroRNAs/metabolismo , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/genética , Animais , Ácido Quenodesoxicólico/análogos & derivados , Ácido Quenodesoxicólico/farmacologia , Colestase/tratamento farmacológico , Colestase/genética , Colestase/patologia , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Knockout , MicroRNAs/genética , Correpressor 1 de Receptor Nuclear/genética , Correpressor 1 de Receptor Nuclear/metabolismo
3.
J Pediatr Gastroenterol Nutr ; 73(2): 169-177, 2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-34016879

RESUMO

OBJECTIVES: To advance our understanding of monogenic forms of intrahepatic cholestasis. METHODS: Analyses included participants with pathogenic biallelic mutations in adenosine triphosphate (ATP)-binding cassette subfamily B member 11 (ABCB11) (bile salt export pump; BSEP) or adenosine triphosphatase (ATPase) phospholipid transporting 8B1 (ATP8B1) (familial intrahepatic cholestasis; FIC1), or those with monoallelic or biallelic mutations in adenosine triphosphate (ATP)-binding cassette subfamily B member 4 (ABCB4) (multidrug resistance; MDR3), prospectively enrolled in the Longitudinal Study of Genetic Causes of Intrahepatic Cholestasis (LOGIC; NCT00571272) between November 2007 and December 2013. Summary statistics were calculated to describe baseline demographics, history, anthropometrics, laboratory values, and mutation data. RESULTS: Ninety-eight participants with FIC1 (n = 26), BSEP (n = 53, including 8 with biallelic truncating mutations [severe] and 10 with p.E297G or p.D482G [mild]), or MDR3 (n = 19, including four monoallelic) deficiency were analyzed. Thirty-five had a surgical interruption of the enterohepatic circulation (sEHC), including 10 who underwent liver transplant (LT) after sEHC. Onset of symptoms occurred by age 2 years in most with FIC1 and BSEP deficiency, but was later and more variable for MDR3. Pruritus was nearly universal in FIC1 and BSEP deficiency. In participants with native liver, failure to thrive was common in FIC1 deficiency, high ALT was common in BSEP deficiency, and thrombocytopenia was common in MDR3 deficiency. sEHC was successful after more than 1 year in 7 of 19 participants with FIC1 and BSEP deficiency. History of LT was most common in BSEP deficiency. Of 102 mutations identified, 43 were not previously reported. CONCLUSIONS: In this cohort, BSEP deficiency appears to be correlated with a more severe disease course. Genotype-phenotype correlations in these diseases are not straightforward and will require the study of larger cohorts.


Assuntos
Colestase Intra-Hepática , Colestase , Transportadores de Cassetes de Ligação de ATP/genética , Criança , Pré-Escolar , Colestase/genética , Colestase Intra-Hepática/genética , Humanos , Estudos Longitudinais , Mutação
4.
J Pediatr ; 202: 179-185.e4, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30244988

RESUMO

OBJECTIVE: To investigate the impact of corticosteroid therapy on the growth of participants in the Steroids in Biliary Atresia Randomized Trial (START) conducted through the Childhood Liver Disease Research Network. The primary analysis in START indicated that steroids did not have a beneficial effect on drainage in a cohort of infants with biliary atresia. We hypothesized that steroids would have a detrimental effect on growth in these infants. STUDY DESIGN: A total of 140 infants were enrolled in START, with 70 randomized to each treatment arm: steroid and placebo. Length, weight, and head circumference were obtained at baseline and follow-up visits to 24 months of age. RESULTS: Patients treated with steroids had significantly lower length and head circumference z scores during the first 3 months post-hepatoportoenterostomy (HPE), and significantly lower weight until 12 months. Growth trajectories in the steroid and placebo arms differed significantly for length (P < .0001), weight (P = .009), and head circumference (P < .0001) with the largest impact noted for those with successful HPE. Growth trajectory for head circumference was significantly lower in patients treated with steroids irrespective of HPE status, but recovered during the second 6 months of life. CONCLUSIONS: Steroid therapy following HPE in patients with biliary atresia is associated with impaired length, weight, and head circumference growth trajectories for at least 6 months post-HPE, especially impacting infants with successful bile drainage. TRIAL REGISTRATION: ClinicalTrials.gov: NCT00294684.


Assuntos
Corticosteroides/efeitos adversos , Atresia Biliar/tratamento farmacológico , Atresia Biliar/cirurgia , Insuficiência de Crescimento/induzido quimicamente , Sarcopenia/induzido quimicamente , Corticosteroides/uso terapêutico , Atresia Biliar/mortalidade , Peso Corporal/efeitos dos fármacos , Cefalometria/métodos , Desenvolvimento Infantil/efeitos dos fármacos , Desenvolvimento Infantil/fisiologia , Pré-Escolar , Método Duplo-Cego , Insuficiência de Crescimento/epidemiologia , Insuficiência de Crescimento/fisiopatologia , Feminino , Seguimentos , Humanos , Lactente , Masculino , Monitorização Fisiológica/métodos , Portoenterostomia Hepática/métodos , Portoenterostomia Hepática/mortalidade , Cuidados Pós-Operatórios/métodos , Estudos Prospectivos , Valores de Referência , Medição de Risco , Sarcopenia/epidemiologia , Sarcopenia/fisiopatologia , Fatores de Tempo , Resultado do Tratamento
5.
Hum Mol Genet ; 24(10): 2724-32, 2015 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-25631877

RESUMO

The control of transcription is regulated through the well-coordinated spatial and temporal interactions between distal genomic regulatory elements required for specialized cell-type and developmental gene expression programs. With recent findings CFTR has served as a model to understand the principles that govern genome-wide and topological organization of distal intra-chromosomal contacts as it relates to transcriptional control. This is due to the extensive characterization of the DNase hypersensitivity sites, modification of chromatin, transcription factor binding sites and the arrangement of these sites in CFTR consistent with the restrictive expression in epithelial cell types. Here, we identified CHD6 from a screen among several chromatin-remodeling proteins as a putative epigenetic modulator of CFTR expression. Moreover, our findings of CTCF interactions with CHD6 are consistent with the role described previously for CTCF in CFTR regulation. Our results now reveal that the CHD6 protein lies within the infrastructure of multiple transcriptional complexes, such as the FACT, PBAF, PAF1C, Mediator, SMC/Cohesion and MLL complexes. This model underlies the fundamental role CHD6 facilitates by tethering cis-acting regulatory elements of CFTR in proximity to these multi-subunit transcriptional protein complexes. Finally, we indicate that CHD6 structurally coordinates a three-dimensional stricture between intragenic elements of CFTR bound by several cell-type specific transcription factors, such as CDX2, SOX18, HNF4α and HNF1α. Therefore, our results reveal new insights into the epigenetic regulation of CFTR expression, whereas the manipulation of CFTR gene topology could be considered for treating specific indications of cystic fibrosis and/or pancreatitis.


Assuntos
Cromatina/química , Regulador de Condutância Transmembrana em Fibrose Cística/genética , DNA Helicases/metabolismo , Loci Gênicos , Proteínas do Tecido Nervoso/metabolismo , Elementos Reguladores de Transcrição , Epigênese Genética , Humanos , Conformação de Ácido Nucleico , Sequências Reguladoras de Ácido Nucleico , Fatores de Transcrição/metabolismo
6.
Am J Physiol Gastrointest Liver Physiol ; 310(8): G618-28, 2016 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-26867564

RESUMO

In this study we identified the mechanisms underlying the inhibitory effects of NF-κB on the expression of genes encoding multiple liver transport proteins. Well-conserved NF-κB binding sites were found in the promoters of farnesoid X receptor (FXR)-target genes. An electromobility shift assay (EMSA) demonstrated the specific interaction between the NF-κB p65 protein and a (32)P-labeled BSEP NF-κB response element (NF-κBE). Chromatin immunoprecipitation (ChIP) analysis confirmed binding of NF-κB p65 to the BSEP locus but not the FXRE in vitro. NF-κB p65 overexpression in Huh-7 cells markedly repressed FXR/RXR transactivation of the BSEP, ABCG5/G8, MRP2, and FXR promoters, which was totally reversed by expression of the IκBα super-repressor. NF-κB interacted directly with FXR on coimmunoprecipitation, suggesting another level for the inhibitory effects of NF-κB on FXR-target genes. In vivo ChIP analysis with liver nuclei obtained from mice after 3 days of common bile duct ligation (BDL) or 6 h post-lipopolysaccharide (LPS) injection showed a markedly increased recruitment of NF-κB p65 to the Bsep promoter compared with controls. There was also increased recruitment of the corepressor silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) and histone deacetylase (HDAC)3 and HDAC2 to the NF-κB sites. We also found that NF-κB p65 was recruited to NF-κB binding sites in the promoters of organic solute transporter, OSTα and OSTß, and unexpectedly activated rather than repressed gene expression. In mouse liver after BDL NF-κB recruitment to Ostα and Ostß promoters was associated with increased binding of the potent coactivator cAMP response element binding protein (CREB)-binding protein (CBP)/p300 to the NF-κBE and depletion of CBP/p300 at the FXR element. Overall, these studies demonstrate a novel role for NF-κB in adaptation to obstructive and LPS-induced cholestasis acting through recruitment to specific NF-κB binding sites in the promoters of FXR-target genes and possibly through direct interaction with FXR.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Fígado/metabolismo , Elementos de Resposta , Fator de Transcrição RelA/metabolismo , Ativação Transcricional , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Linhagem Celular Tumoral , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Correpressor 2 de Receptor Nuclear/genética , Correpressor 2 de Receptor Nuclear/metabolismo , Ligação Proteica
7.
J Biol Chem ; 288(19): 13850-62, 2013 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-23546875

RESUMO

BACKGROUND: Small ubiquitin-like modifiers (SUMO) are covalently conjugated to other proteins including nuclear receptors leading to modification of various cellular processes. RESULTS: Ligand-dependent SUMOylation of farnesoid X receptor (FXR) negatively regulates the expression of its target genes. CONCLUSION: SUMO modification attenuates the capacity of FXR to function as a transcriptional activator. SIGNIFICANCE: Defining post-translation modification of FXR bySUMOis important to understanding how this nuclear receptor functions in health and disease. The farnesoid X receptor (FXR) belongs to a family of ligand-activated transcription factors that regulate many aspects of metabolism including bile acid homeostasis. Here we show that FXR is covalently modified by the small ubiquitin-like modifier (Sumo1), an important regulator of cell signaling and transcription. Well conserved consensus sites at lysine 122 and 275 in the AF-1 and ligand binding domains, respectively, of FXR were subject to SUMOylation in vitro and in vivo. Chromatin immunoprecipitation (ChIP) analysis showed that Sumo1 was recruited to the bile salt export pump (BSEP), the small heterodimer partner (SHP), and the OSTα-OSTß organic solute transporter loci in a ligand-dependent fashion. Sequential chromatin immunoprecipitation (ChIP-ReChIP) verified the concurrent binding of FXR and Sumo1 to the BSEP and SHP promoters. Overexpression of Sumo1 markedly decreased binding and/or recruitment of FXR to the BSEP and SHP promoters on ChIP-ReChIP. SUMOylation did not have an apparent effect on nuclear localization of FXR. Expression of Sumo1 markedly inhibited the ligand-dependent, transactivation of BSEP and SHP promoters by FXR/retinoid X receptor α (RXRα) in HepG2 cells. In contrast, mutations that abolished SUMOylation of FXR or siRNA knockdown of Sumo1 expression augmented the transactivation of BSEP and SHP promoters by FXR. Pathways for SUMOylation were significantly altered during obstructive cholestasis with differential Sumo1 recruitment to the promoters of FXR target genes. In conclusion, FXR is subject to SUMOylation that regulates its capacity to transactivate its target genes in normal liver and during obstructive cholestasis.


Assuntos
Regulação da Expressão Gênica , Receptores Citoplasmáticos e Nucleares/metabolismo , Sumoilação , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Motivos de Aminoácidos , Substituição de Aminoácidos , Animais , Colestase/metabolismo , Cromatina/metabolismo , Sequência Consenso , Técnicas de Silenciamento de Genes , Células Hep G2 , Humanos , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mutagênese Sítio-Dirigida , Regiões Promotoras Genéticas , Ligação Proteica , Processamento de Proteína Pós-Traducional , Transporte Proteico , RNA Interferente Pequeno/genética , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/genética , Proteína SUMO-1/genética , Proteína SUMO-1/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Transcrição Gênica
8.
Am J Physiol Gastrointest Liver Physiol ; 306(2): G154-62, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24264050

RESUMO

The organic solute transporter-α/ß (OSTα/ß) is a heteromeric transporter that is essential for bile acid and sterol disposition and for the enterohepatic circulation. To better understand the mechanism underlying OST gene regulation, the effects of retinoic acid (RA) on OSTα/ß gene expression were investigated. The results show a dose-dependent induction of OSTß but not OSTα expression in both Huh7 and HepG2 cells by RA treatment. A novel functional RA receptor response element (RARE; so-called DR5) in the promoter of OSTß gene was identified. The interaction of RARα/RXRα with the RARE was verified by electrophoretic mobility shift and chromatin immunoprecipitation assays and its functional importance by hOSTß promoter activation in luciferase reporter assays. The studies demonstrated that the RARE is also a constitutive androstane receptor (CAR) binding site for OSTß gene regulation. These results suggest that OSTß is a target of both FXR-mediated (by binding to IR-1 element) and RARα- and CAR-mediated (by binding to DR5 element) gene regulation pathways. In summary, this study has uncovered a novel RARE (DR5) element in the promoter of OSTß that binds RARα or CAR heterodimerized with RXRα and appears to function synergistically with the IR-1 element to provide maximal induction of OSTß in response to RA. These findings demonstrate a role for RARα and CAR in controlling OSTß expression levels.


Assuntos
Proteínas de Membrana Transportadoras/biossíntese , Receptores do Ácido Retinoico/fisiologia , Sequência de Bases , Western Blotting , Linhagem Celular Tumoral , Colestase/metabolismo , Colesterol/metabolismo , Imunoprecipitação da Cromatina , Ensaio de Desvio de Mobilidade Eletroforética , Genes Reporter , Humanos , Proteínas de Membrana Transportadoras/genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , RNA/biossíntese , RNA/genética , RNA Interferente Pequeno/farmacologia , Reação em Cadeia da Polimerase em Tempo Real , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores Citoplasmáticos e Nucleares/fisiologia , Elementos de Resposta/fisiologia , Receptor alfa de Ácido Retinoico , Receptor X Retinoide alfa/metabolismo , Transfecção , Tretinoína/farmacologia , Xenobióticos/metabolismo
9.
JAMA ; 311(17): 1750-9, 2014 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-24794368

RESUMO

IMPORTANCE: Biliary atresia is the most common cause of end-stage liver disease in children. Controversy exists as to whether use of steroids after hepatoportoenterostomy improves clinical outcome. OBJECTIVE: To determine whether the addition of high-dose corticosteroids after hepatoportoenterostomy is superior to surgery alone in improving biliary drainage and survival with the native liver. DESIGN, SETTING, AND PATIENTS: The multicenter, double-blind Steroids in Biliary Atresia Randomized Trial (START) was conducted in 140 infants (mean age, 2.3 months) between September 2005 and February 2011 in the United States; follow-up ended in January 2013. INTERVENTIONS: Participants were randomized to receive intravenous methylprednisolone (4 mg/kg/d for 2 weeks) and oral prednisolone (2 mg/kg/d for 2 weeks) followed by a tapering protocol for 9 weeks (n = 70) or placebo (n = 70) initiated within 72 hours of hepatoportoenterostomy. MAIN OUTCOMES AND MEASURES: The primary end point (powered to detect a 25% absolute treatment difference) was the percentage of participants with a serum total bilirubin level of less than 1.5 mg/dL with his/her native liver at 6 months posthepatoportoenterostomy. Secondary outcomes included survival with native liver at 24 months of age and serious adverse events. RESULTS: The proportion of participants with improved bile drainage was not statistically significantly improved by steroids at 6 months posthepatoportoenterostomy (58.6% [41/70] of steroids group vs 48.6% [34/70] of placebo group; adjusted relative risk, 1.14 [95% CI, 0.83 to 1.57]; P = .43). The adjusted absolute risk difference was 8.7% (95% CI, -10.4% to 27.7%). Transplant-free survival was 58.7% in the steroids group vs 59.4% in the placebo group (adjusted hazard ratio, 1.0 [95% CI, 0.6 to 1.8]; P = .99) at 24 months of age. The percentage of participants with serious adverse events was 81.4% [57/70] of the steroids group and 80.0% [56/70] of the placebo group (P > .99); however, participants receiving steroids had an earlier time of onset of their first serious adverse event by 30 days posthepatoportoenterostomy (37.2% [95% CI, 26.9% to 50.0%] of steroids group vs 19.0% [95% CI, 11.5% to 30.4%] of placebo group; P = .008). CONCLUSIONS AND RELEVANCE: Among infants with biliary atresia who have undergone hepatoportoenterostomy, high-dose steroid therapy following surgery did not result in statistically significant treatment differences in bile drainage at 6 months, although a small clinical benefit could not be excluded. Steroid treatment was associated with earlier onset of serious adverse events in children with biliary atresia. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00294684.


Assuntos
Corticosteroides/administração & dosagem , Atresia Biliar/tratamento farmacológico , Atresia Biliar/cirurgia , Metilprednisolona/administração & dosagem , Portoenterostomia Hepática , Prednisolona/administração & dosagem , Administração Oral , Corticosteroides/efeitos adversos , Bilirrubina/sangue , Método Duplo-Cego , Drenagem/métodos , Feminino , Humanos , Lactente , Infusões Intravenosas , Masculino , Metilprednisolona/efeitos adversos , Prednisolona/efeitos adversos , Análise de Sobrevida , Resultado do Tratamento
10.
Mol Pharmacol ; 83(5): 1078-86, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23462506

RESUMO

Base amino acid lysine residues play an important role in regulation of nuclear receptors [e.g., farnesyl X receptor (FXR)], leading to enhanced or suppressed biologic activity. To understand the molecular mechanisms and the subsequent effects in modulating FXR functions in diverse biologic processes, we individually replaced eight highly conserved lysine residues of human FXR (hFXR) with arginine. The effects of each mutated FXR on target gene activation, subcellular localization, protein-protein association, and protein-DNA interaction were investigated. Results demonstrated that K122R, K210R, K339R, and K460R mutants of hFXR significantly impaired target gene [organic solute transporter α/ß and bile salt export pump (BSEP)] promoter reporter activity in a ligand-dependent fashion. None of the four mutants affected the nuclear localization of FXR. Protein interaction studies show that K210R slightly but significantly decreased FXR/retinoid X receptor (RXR) binding affinity but enhanced the interaction of FXR with lysine methyltransferase Set7/9 by ∼21%. K460R decreased the FXR interaction with Set7/9 by ∼45% but had no significant effects on interaction with RXR. Electrophoretic mobility shift assays demonstrated that hFXR-K210R and -K339R reduced the protein-DNA (IR1 element at hBSEP promoter) binding affinity by ∼80 and ∼90%, respectively. Computational-based protein modeling studies were consistent with these results and provided further insights into the potential underlying mechanisms responsible for these results. In conclusion, four highly conserved lysine residues of hFXR, K122, K210, K339, and K460, have been identified that play a critical role in FXR target gene regulation and molecular interaction (protein-protein and protein-DNA).


Assuntos
Lisina/genética , Lisina/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Ativação Transcricional/genética , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Sequência de Aminoácidos , Arginina/genética , Arginina/metabolismo , Células CACO-2 , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Inibidores de Histona Desacetilases/farmacologia , Humanos , Ligantes , Metiltransferases/genética , Metiltransferases/metabolismo , Dados de Sequência Molecular , Mutação/efeitos dos fármacos , Mutação/genética , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Domínios e Motivos de Interação entre Proteínas/genética , Receptores X de Retinoides/genética , Receptores X de Retinoides/metabolismo , Ativação Transcricional/efeitos dos fármacos
11.
Am J Physiol Gastrointest Liver Physiol ; 302(9): G937-47, 2012 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-22345554

RESUMO

The farnesoid X receptor (FXR) is a ligand (bile acid)-dependent nuclear receptor that regulates target genes involved in every aspect of bile acid homeostasis. Upon binding of ligand, FXR recruits an array of coactivators and associated proteins, some of which have intrinsic enzymatic activity that modify histones or even components of the transcriptional complex. In this study, we show chromatin occupancy by the Set7/9 methyltransferase at the FXR response element (FXRE) and direct methylation of FXR in vivo and in vitro at lysine 206. siRNA depletion of Set7/9 in the Huh-7 liver cell line decreased endogenous mRNAs of the FXR target genes, the short heterodimer partner (SHP) and bile salt export pump (BSEP). Mutation of the methylation site at K206 of FXR to an arginine prevented methylation by Set7/9. A pan-methyllysine antibody recognized the wild-type FXR but not the K206R mutant form. An electromobility shift assay showed that methylation by Set7/9 enhanced binding of FXR/retinoic X receptor-α to the FXRE. Interaction between hinge domain of FXR (containing K206) and Set7/9 was confirmed by coimmunoprecipitation, GST pull down, and mammalian two-hybrid experiments. Set7/9 overexpression in Huh-7 cells significantly enhanced transactivation of the SHP and BSEP promoters in a ligand-dependent fashion by wild-type FXR but not the K206R mutant FXR. A Set7/9 mutant deficient in methyltransferase activity was also not effective in increasing transactivation of the BSEP promoter. These studies demonstrate that posttranslational methylation of FXR by Set7/9 contributes to the transcriptional activation of FXR-target genes.


Assuntos
Hepatócitos/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Rim/metabolismo , Ativação Transcricional/fisiologia , Animais , Linhagem Celular , Regulação da Expressão Gênica/fisiologia , Haplorrinos , Histona-Lisina N-Metiltransferase/genética , Humanos , Metilação , Regiões Promotoras Genéticas/genética , Ligação Proteica , Receptores Citoplasmáticos e Nucleares
12.
JPEN J Parenter Enteral Nutr ; 46(5): 1096-1106, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-34664730

RESUMO

BACKGROUND: We have recently reported a mouse model of PN-associated cholestasis (PNAC) in which combining intestinal inflammation and PN infusion results in cholestasis, hepatic macrophage activation, and transcriptional suppression of canalicular bile acid, bilirubin and sterol transporters Abcb11, Abcc2 and Abcg5/8. The aim of this study was to examine the role of TNFα in promoting PNAC in mice. METHODS: First, recombinant TNFα was administered to mice as well as in hepatocyte cell culture. Second, Tnfr1/2KO or wild-type (WT) mice were exposed to dextran sulfate sodium (DSS) for 4 days followed by soy-oil lipid emulsion-based PN infusion through a central venous catheter for 14 days (DSS-PN). Finally, WT/DSS-PN mice were also infused with infliximab at 10 mg/kg on days 3 and 10 of PN. PNAC was defined by increased serum aspartate aminotransferase, alanine aminotransferase, total bile acids, and bilirubin. RESULTS: Intraperitoneal injection of TNFα into WT mice or TNFα treatment of Huh7 hepatocarcinoma cells and primary mouse hepatocytes suppressed messenger RNA (mRNA) transcription of bile (Abcb11, Abcc2]) and sterol transporters (Abcg5/8) and their regulators Nr1h3 and Nr1h4. DSS-PN mice with PNAC had increased hepatic TNFα mRNA expression and significant reduction of mRNA expression of Abcb11, Abcc2, Abcg5/8, Nr1h3, and Nr1h4. In contrast, PNAC development was prevented and mRNA expression normalized in both Tnfr1/2KO /DSS-PN mice and DSS-PN mice treated with infliximab. CONCLUSIONS: TNFα is a key mediator in the pathogenesis of PNAC through suppression of hepatocyte Abcb11, Abcc2, and Abcg5/8. Pharmacologic targeting of TNFα as a therapeutic strategy for PNAC thus deserves further investigation.


Assuntos
Colestase , Fator de Necrose Tumoral alfa , Animais , Ácidos e Sais Biliares , Bilirrubina , Colestase/etiologia , Infliximab , Camundongos , Nutrição Parenteral , RNA Mensageiro , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Esteróis
13.
Semin Liver Dis ; 31(1): 3-10, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21344347

RESUMO

Progressive familial intrahepatic cholestatic diseases encompass a group of autosomal recessive hereditary diseases, which usually present in infancy or childhood, with cholestasis of hepatocellular origin. The currently preferred nomenclature for the three PFIC disorders that have been characterized to date is FIC1 deficiency, BSEP deficiency, and MDR3 deficiency, relating to mutations in the specific genes involved in bile acid formation and transport. Since the first description of these diseases, extensive clinical, biochemical, and molecular studies have increased our understanding of the features specific to each one of them. This review focuses mainly on the liver histology, summarizing their characteristic pathologic features, the correlation to specific genotypes, and complications arising with disease progression.


Assuntos
Fígado/patologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/deficiência , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Adenosina Trifosfatases/deficiência , Adenosina Trifosfatases/genética , Colestase Intra-Hepática/classificação , Colestase Intra-Hepática/genética , Colestase Intra-Hepática/patologia , Progressão da Doença , Predisposição Genética para Doença , Hereditariedade , Humanos , Fenótipo , Prognóstico , Terminologia como Assunto
14.
Am J Physiol Gastrointest Liver Physiol ; 300(5): G771-81, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21330447

RESUMO

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.


Assuntos
Proteínas de Transporte/genética , Colestase/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Glicoproteínas de Membrana/genética , Coativadores de Receptor Nuclear/metabolismo , Receptores Citoplasmáticos e Nucleares/fisiologia , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/biossíntese , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Ductos Biliares/fisiologia , Células Cultivadas , Colestase/genética , Regulação para Baixo , Glutationa Transferase/metabolismo , Histona-Lisina N-Metiltransferase/genética , Humanos , Imunoprecipitação , Ligadura , Metilação , Camundongos , Coativadores de Receptor Nuclear/genética , Transportadores de Ânions Orgânicos Dependentes de Sódio/biossíntese , Transportadores de Ânions Orgânicos Dependentes de Sódio/genética , Plasmídeos/genética , RNA Interferente Pequeno/genética , Receptores Citoplasmáticos e Nucleares/genética , Simportadores/biossíntese , Simportadores/genética
16.
Clin Transplant ; 25(6): 849-55, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21175846

RESUMO

BACKGROUND: Hepatopulmonary syndrome (HPS) is the association of liver disease, hypoxemia, and intrapulmonary vascular dilatations. There are little data on the management of HPS in children other than conventional orthotopic liver transplantation (OLT). AIMS: To describe the patient characteristics, mode of diagnosis, treatment, and outcomes of children with HPS at our center. METHODS: Retrospective review of patients diagnosed with HPS between 1997 and 2007 after IRB approval. RESULTS: There were 10 patients, six females; median age at diagnosis of HPS was 12 yr. Six with cirrhosis underwent OLT and had subsequent resolution of HPS and are stable at last follow-up. Of the remaining four, two had cirrhosis. HPS resolved without conventional OLT in the following four patients: hepatitis C after antiviral treatment, biliary atresia with portal hypertension after transjugular intrahepatic portosystemic shunting, Abernethy syndrome after auxiliary partial OLT, and in a child with splenic vein thrombosis after splenectomy. CONCLUSIONS: Our series shows resolution of HPS in all patients and 100% survival after conventional OLT. Four children had resolution of HPS after surgical or medical treatments other than conventional OLT. Careful review of clinical status and underlying pathophysiology and anatomy at diagnosis of HPS should inform treatment decisions.


Assuntos
Síndrome Hepatopulmonar/mortalidade , Síndrome Hepatopulmonar/terapia , Transplante de Fígado/estatística & dados numéricos , Adolescente , Criança , Pré-Escolar , Feminino , Seguimentos , Síndrome Hepatopulmonar/diagnóstico , Humanos , Masculino , Estudos Retrospectivos , Taxa de Sobrevida , Resultado do Tratamento
17.
Isr Med Assoc J ; 13(9): 542-7, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21991714

RESUMO

BACKGROUND: Primary liver masses in children may require intervention because of symptoms or concern about malignant transformation. OBJECTIVES: To review the management and outcome of benign liver masses in children. METHODS: We conducted a retrospective chart review of children with liver masses referred to our institution during the period 1997-2009. RESULTS: Benign liver masses were identified in 53 children. Sixteen of these children (30%) had hemangioma/infantile hepatic hemangioendothelioma (IHH) and 15 (28%) had focal nodular hyperplasia. The remainder had 6 cysts, 4 hamartomas, 3 nodular regenerative hyperplasia, 2 adenomas, 2 vascular malformations, and one each of polyarteritis nodosa, granuloma, hepatic hematoma, lymphangioma, and infarction. Median age at presentation was 6 years, and 30 (57%) were female. Masses were initially noticed on imaging studies performed for unrelated symptoms in 33 children (62%), laboratory abnormalities consistent with liver disease in 11 (21%), and palpable abdominal masses in 9 (17%). Diagnosis was made based on characteristic radiographic findings in 31 (58%), but histopathological examination was required for the remaining 22 (42%). Of the 53 children, 27 (51%) were under observation while 17 (32%) had masses resected. Medications targeting masses were used in 9 (17%) and liver transplantation was performed in 4 (8%). The only death (2%) occurred in a child with multifocal IHH unresponsive to medical management and prior to liver transplant availability. CONCLUSIONS: IHH and focal nodular hyperplasia were the most common lesions. The majority of benign lesions were found incidentally and diagnosed radiologically. Expectant management was sufficient in most children after diagnosis, although surgical intervention including liver transplant was occasionally necessary.


Assuntos
Neoplasias Hepáticas/diagnóstico , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Hiperplasia Nodular Focal do Fígado/diagnóstico , Hiperplasia Nodular Focal do Fígado/tratamento farmacológico , Hiperplasia Nodular Focal do Fígado/cirurgia , Hemangioendotelioma/diagnóstico , Hemangioendotelioma/tratamento farmacológico , Hemangioendotelioma/cirurgia , Hemangioma/diagnóstico , Hemangioma/tratamento farmacológico , Hemangioma/cirurgia , Humanos , Achados Incidentais , Lactente , Recém-Nascido , Fígado/patologia , Fígado/cirurgia , Hepatopatias/diagnóstico , Hepatopatias/tratamento farmacológico , Hepatopatias/cirurgia , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/cirurgia , Masculino , Estudos Retrospectivos , Resultado do Tratamento , Adulto Jovem
19.
Hepatology ; 48(6): 1896-905, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18668687

RESUMO

UNLABELLED: Prior loss-of-function analyses revealed that ATPase class I type 8B member 1 [familial intrahepatic cholestasis 1 (FIC1)] posttranslationally activated the farnesoid X receptor (FXR). Mechanisms underlying this regulation were examined by gain-of-function studies in UPS cells, which lack endogenous FIC1 expression. FXR function was assayed in response to wild-type and mutated FIC1 expression constructs with a human bile salt export pump (BSEP) promoter and a variety of cellular localization techniques. FIC1 overexpression led to enhanced phosphorylation and nuclear localization of FXR that was associated with FXR-dependent activation of the BSEP promoter. The FIC1 effect was lost after mutation of the FXR response element in the BSEP promoter. Despite similar levels of FIC1 protein expression, Byler disease FIC1 mutants did not activate BSEP, whereas benign recurrent intrahepatic cholestasis mutants partially activated BSEP. The FIC1 effect was dependent on the presence of the FXR ligand, chenodeoxycholic acid. The effect of FIC1 on FXR phosphorylation and nuclear localization and its effects on BSEP promoter activity could be blocked with protein kinase C zeta (PKC zeta) inhibitors (pseudosubstrate or small interfering RNA silencing). Recombinant PKC zeta directly phosphorylated immunoprecipitated FXR. The mutation of threonine 442 of FXR to alanine yielded a dominant negative protein, whereas the phosphomimetic conversion to glutamate resulted in FXR with enhanced activity and nuclear localization. Inhibition of PKC zeta in Caco-2 cells resulted in activation of the human apical sodium-dependent bile acid transporter promoter. CONCLUSION: These results demonstrate that FIC1 signals to FXR via PKC zeta. FIC1-related liver disease is likely related to downstream effects of FXR on bile acid homeostasis. Benign recurrent intrahepatic cholestasis emanates from a partially functional FIC1 protein. Phosphorylation of FXR is an important mechanism for regulating its activity.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteína Quinase C/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Transdução de Sinais/fisiologia , Fatores de Transcrição/metabolismo , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Ácidos e Sais Biliares/metabolismo , Células CACO-2 , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Proteínas de Fluorescência Verde , Haplorrinos , Homeostase , Humanos , Rim/citologia , Rim/embriologia , Fosforilação , Plasmídeos , Proteína Quinase C/antagonistas & inibidores , Transfecção
20.
Hepatol Commun ; 3(12): 1674-1686, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31832574

RESUMO

Adenosine triphosphate-binding cassette subfamily C member 2 (ABCC2/Abcc2) is critically important to biliary excretion of many endobiotic and xenobiotic compounds, and is a major driving force for bile acid-independent bile flow. Abcc2 expression is reduced at the messenger RNA (mRNA) and protein levels in various forms of experimental cholestasis. In a microRNA (miRNA) screen of mouse liver after biliary obstruction, we found that miRNA let7a-5p was significantly up-regulated approximately 4-fold. Similarly, ABCC2 mRNA was depleted and miRNA let7a-5p was elevated over 4-fold in livers of children with biliary atresia compared with normal livers. In silico analysis predicted that let7a-5p would target the 3' untranslated region (3' UTR) of ABCC2/Abcc2 RNA. The objective of this study was to determine whether let7a-5p contributes to the depletion of ABCC2/Abcc2 in cholestasis. To demonstrate the functional importance of miRNA let7a-5p in regulating the expression of ABCC2, co-transfection of a let7a-5p mimic and an ABCC2-3' UTR luciferase construct into Huh-7 cells led to a marked inhibition of luciferase activity by about 60%-70% compared with controls, which was reversed by a let7a-5p mimic inhibitor. Expression of this mimic led to a significant decrease in endogenous ABCC2 mRNA and protein levels in a Huh-7 liver cell line, which could be blocked by expression of a let7a-5p mimic inhibitor. Injection of a lentivirus let7a-5p inhibitor into normal mouse liver or into mouse liver after common bile duct ligation led to a significant increase in endogenous Abcc2 mRNA and protein levels and a depletion of let7a-5p mRNA levels compared with untreated, saline-injected livers or livers treated with an inactive lentivirus control. Conclusion: These studies demonstrate that miR-let7a-5p is involved in regulating ABCC2/Abcc2 expression, and is aberrantly up-regulated in obstructive cholestasis.

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