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1.
Gut ; 69(2): 231-242, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31068366

RESUMO

OBJECTIVE: Gastric cancer (GC) is a leading cause of cancer mortality. Previous studies have shown that hepatocyte nuclear factor-4α (HNF4α) is specifically overexpressed in GC and functionally required for GC development. In this study, we investigated, on a genome-wide scale, target genes of HNF4α and oncogenic pathways driven by HNF4α and HNF4α target genes. DESIGN: We performed HNF4α chromatin immunoprecipitation followed by sequencing across multiple GC cell lines, integrating HNF4α occupancy data with (epi)genomic and transcriptome data of primary GCs to define HNF4α target genes of in vitro and in vivo relevance. To investigate mechanistic roles of HNF4α and HNF4α targets, we performed cancer metabolic measurements, drug treatments and functional assays including murine xenograft experiments. RESULTS: Gene expression analysis across 19 tumour types revealed HNF4α to be specifically upregulated in GCs. Unbiased pathway analysis revealed organic acid metabolism as the top HNF4α-regulated pathway, orthogonally supported by metabolomic analysis. Isocitrate dehydrogenase 1 (IDH1) emerged as a convergent HNF4α direct target gene regulating GC metabolism. We show that wild-type IDH1 is essential for GC cell survival, and that certain GC cells can be targeted by IDH1 inhibitors. CONCLUSIONS: Our results highlight a role for HNF4α in sustaining GC oncogenic metabolism, through the regulation of IDH1. Drugs targeting wild-type IDH1 may thus have clinical utility in GCs exhibiting HNF4α overexpression, expanding the role of IDH1 in cancer beyond IDH1/2 mutated malignancies.


Assuntos
Fator 4 Nuclear de Hepatócito/genética , Isocitrato Desidrogenase/metabolismo , Neoplasias Gástricas/genética , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Feminino , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Marcação de Genes/métodos , Fator 4 Nuclear de Hepatócito/metabolismo , Humanos , Isocitrato Desidrogenase/antagonistas & inibidores , Camundongos Endogâmicos NOD , Terapia de Alvo Molecular/métodos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Regiões Promotoras Genéticas/genética , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Regulação para Cima/genética , Ensaios Antitumorais Modelo de Xenoenxerto
2.
Artigo em Inglês | MEDLINE | ID: mdl-31525459

RESUMO

As the first marine teleost demonstrated to biosynthesize long-chain polyunsaturated fatty acids (LC-PUFAs) from C18 precursors such as linoleic acid (LOA, 18:2n-6) and α-linolenic acid (ALA, 18:3n-3), the rabbitfish (Siganus canaliculatus) contains the complete enzymatic system for LC-PUFA biosynthesis, including Δ6/Δ5 fatty acid desaturase (Fad), Δ4 Fad, and elongase 5 (Elovl5). Previously, our group demonstrated that hepatocyte nuclear factor 4α (Hnf4α) is a transcription factor (TF) for rabbitfish Δ4 fad and elovl5, and interacts with the core promoter of Δ6/Δ5 fad. To fully clarify the role of Hnf4α in the regulation of LC-PUFA biosynthesis, the present study aimed to explore the regulatory role of Hnf4α on Δ6/Δ5 fad gene expression. First, Hnf4α overexpression and agonist assays identified the Hnf4α response region in the Δ6/Δ5 fad core promoter as -456 bp to +51 bp. Bioinformatic analysis predicted four potential Hnf4α binding elements in the core promoter, which were confirmed by site-directed mutation and functional assays in a dual luciferase assay system. Moreover, the mRNA expression levels of hnf4α, Δ6/Δ5 fad, and Δ4 fad were significantly increased in the S. canaliculatus hepatocyte line (SCHL) cells after treatment with Hnf4α agonists (Alverine and Benfluorex) or its mRNA overexpression. By contrast, the expression levels of these three genes were markedly decreased after hnf4a small interfering RNA (siRNA) transfection. The results indicated that Hnf4α has a regulatory effect on rabbitfish Δ6/Δ5 fad gene transcription, identifying Hnf4α as a TF of Δ6/Δ5 fad in vertebrates for the first time.


Assuntos
Ácidos Graxos Dessaturases/biossíntese , Proteínas de Peixes/metabolismo , Peixes/metabolismo , Regulação Enzimológica da Expressão Gênica/fisiologia , Fator 4 Nuclear de Hepatócito/metabolismo , Linoleoil-CoA Desaturase/biossíntese , Animais , Ácidos Graxos Dessaturases/genética , Proteínas de Peixes/genética , Peixes/genética , Fator 4 Nuclear de Hepatócito/genética , Linoleoil-CoA Desaturase/genética
3.
J Agric Food Chem ; 67(40): 11119-11128, 2019 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-31525874

RESUMO

Xanthohumol (Xan) is a prenylated chalcone mainly found in hops; it has been demonstrated to function against hypercholesterolemia, hyperlipidemia, and atherosclerosis. In this study, we focused on the hypocholesterolemic effect of Xan on cholesterol uptake and the underlying molecular mechanisms of Xan in human intestinal Caco-2 cells. The microarray data showed that Niemann-Pick C1-like 1 (NPC1L1), an essential transporter for dietary cholesterol absorption, was significantly downregulated in Xan-treated Caco-2 cells. We demonstrated that Xan (10 and 20 µM) suppressed the mRNA and protein expression of NPC1L1 by 0.65 ± 0.12-fold and 0.54 ± 0.15-fold and 0.72 ± 0.04-fold and 0.44 ± 0.12-fold, respectively, compared to that of the vehicle-treated Caco-2 cells. Moreover, Xan (10 and 20 µM) significantly inhibited cholesterol uptake by approximately 12 and 32% in Caco-2 cells. NPC1L1 promoter activity was significantly suppressed by Xan, and a DNA element within the NPC1L1 promoter involved in Xan-mediated NPC1L1 reduction located between the -120 and -20 positions was identified. Moreover, Xan markedly decreased the mRNA and protein levels of hepatocyte nuclear factor 4α (HNF-4α), a critical activator of NPC1L1 transcription, and subsequently attenuated HNF-4α/NPC1L1 promoter complex formation, resulting in the suppression of NPC1L1 gene expression. Finally, we demonstrated that Xan markedly abolished lovastatin-induced NPC1L1 overexpression in Caco-2 cells. These findings reveal that Xan suppresses NPC1L1 expression via downregulation of HNF-4α and exerts inhibitory effects on cholesterol uptake in the intestinal Caco-2 cells. Our findings suggest Xan could serve as a potential cholesterol-lowering agent and supplement for statin therapy.


Assuntos
Colesterol/metabolismo , Flavonoides/farmacologia , Fator 4 Nuclear de Hepatócito/metabolismo , Proteínas de Membrana/genética , Propiofenonas/farmacologia , Anticolesterolemiantes/farmacologia , Transporte Biológico/efeitos dos fármacos , Células CACO-2 , Regulação para Baixo/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Fator 4 Nuclear de Hepatócito/genética , Humanos , Proteínas de Membrana/metabolismo , Regiões Promotoras Genéticas
4.
Medicine (Baltimore) ; 98(28): e16424, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31305461

RESUMO

Although the changes in DNA methylation are assumed to be due to the association between adverse intrauterine conditions and adult metabolic health, evidence from human studies is rare. Little is known about the changes in DNA methylation present at birth that affect metabolic profiles in childhood. Previous studies have shown that the melanocortin 4 receptor (MC4R) and hepatocyte nuclear factor 4 alpha (HNF4α) genes are associated with obesity and metabolic disorders. Thus, we investigated the associations of the DNA methylation statuses of MC4R and HNF4α in cord blood with metabolic profiles in childhood.We collected data from 90 children 7 to 9 years of age included in the Ewha Birth & Growth Cohort Study in Korea. DNA methylation was analyzed by pyrosequencing. The children were split into 2 groups according to the cutoff triglyceride (TG) levels (<110 and ≥110 mg/dL).The methylation statuses of MC4R and HNF4α at birth were significantly associated with the TG level in childhood (P < .05). It was interesting to note that the methylation statuses of MC4R and HNF4α in cord blood were significantly decreased, whereas childhood body mass index was significantly increased, in children with high TG levels compared with children with low TG levels (P < .05).Our findings show that the methylation statuses of MC4R and HNF4α at birth are associated with metabolic profiles in childhood. These epigenetic modifications occurring in early life may contribute to subsequent metabolic-related disorders. Thus, we suggest that DNA methylation status in cord blood may be predictive of the risk of developing metabolic syndrome.


Assuntos
Metilação de DNA , Fator 4 Nuclear de Hepatócito/genética , Regiões Promotoras Genéticas , Receptor Tipo 4 de Melanocortina/genética , Triglicerídeos/sangue , Índice de Massa Corporal , Criança , Desenvolvimento Infantil , Ilhas de CpG , Epigênese Genética , Feminino , Sangue Fetal/metabolismo , Seguimentos , Fator 4 Nuclear de Hepatócito/metabolismo , Humanos , Estudos Longitudinais , Masculino , Estudos Prospectivos , Receptor Tipo 4 de Melanocortina/metabolismo
5.
Nat Commun ; 10(1): 3126, 2019 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-31311938

RESUMO

Alcoholic hepatitis (AH) is a life-threatening condition characterized by profound hepatocellular dysfunction for which targeted treatments are urgently needed. Identification of molecular drivers is hampered by the lack of suitable animal models. By performing RNA sequencing in livers from patients with different phenotypes of alcohol-related liver disease (ALD), we show that development of AH is characterized by defective activity of liver-enriched transcription factors (LETFs). TGFß1 is a key upstream transcriptome regulator in AH and induces the use of HNF4α P2 promoter in hepatocytes, which results in defective metabolic and synthetic functions. Gene polymorphisms in LETFs including HNF4α are not associated with the development of AH. In contrast, epigenetic studies show that AH livers have profound changes in DNA methylation state and chromatin remodeling, affecting HNF4α-dependent gene expression. We conclude that targeting TGFß1 and epigenetic drivers that modulate HNF4α-dependent gene expression could be beneficial to improve hepatocellular function in patients with AH.


Assuntos
Hepatite Alcoólica/genética , Fator 4 Nuclear de Hepatócito/metabolismo , Hepatócitos/patologia , Fígado/patologia , Fator de Crescimento Transformador beta1/metabolismo , Adulto , Idoso , Animais , Biópsia , Montagem e Desmontagem da Cromatina , Metilação de DNA , Progressão da Doença , Epigênese Genética , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Hepatite Alcoólica/patologia , Fator 4 Nuclear de Hepatócito/genética , Humanos , Fígado/citologia , Masculino , Pessoa de Meia-Idade , Polimorfismo Genético , Análise de Sequência de RNA , Fator de Crescimento Transformador beta1/genética
6.
Oncol Rep ; 42(3): 1066-1074, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31322246

RESUMO

Renal cell carcinoma (RCC) is the most common malignant disease of the kidneys in adults. Patients with metastatic RCC have an unusually poor prognosis and exhibit resistance to all current therapies. Therefore, it is necessary to explore novel molecules involved in the progression of RCC and to identify effective therapeutic targets. Hepatocyte nuclear factor­4α (HNF­4α) serves an important role in hepatocyte differentiation and is involved in the progression of liver cancer; however, the functional role of HNF­4α has not been well established in RCC. The present study reported that HNF­4α expression was markedly downregulated in RCC tissue samples compared with in normal controls by immunohistochemistry and RNA­sequencing analysis. Statistical analysis demonstrated that HNF­4α downregulation was significantly associated with tumor stage, recurrence, metastasis and poor prognosis in patients with RCC. Furthermore, wound­healing and Transwell assays revealed that downregulation of HNF­4α promoted cell migration and invasion by transcriptionally regulating E­cadherin in RCC. Finally, a positive correlation was revealed between HNF­4α expression and E­cadherin expression, and patients with low E­cadherin expression also had a poor prognosis. These findings may provide novel insights into the biological effects of HNF­4α and lay the foundation for the discovery of molecular therapeutic targets in RCC.


Assuntos
Antígenos CD/metabolismo , Biomarcadores Tumorais/metabolismo , Caderinas/metabolismo , Carcinoma de Células Renais/patologia , Movimento Celular , Fator 4 Nuclear de Hepatócito/metabolismo , Neoplasias Renais/secundário , Recidiva Local de Neoplasia/patologia , Idoso , Antígenos CD/genética , Apoptose , Biomarcadores Tumorais/genética , Caderinas/genética , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/metabolismo , Estudos de Casos e Controles , Proliferação de Células , Feminino , Seguimentos , Regulação Neoplásica da Expressão Gênica , Fator 4 Nuclear de Hepatócito/genética , Humanos , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/metabolismo , Prognóstico , Taxa de Sobrevida , Células Tumorais Cultivadas
7.
J Agric Food Chem ; 67(28): 8007-8019, 2019 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-31268702

RESUMO

Cow and human milk have been reported to contain dioxins ranging from 0.023 to 26.46 and 0.88 to 19 pg/g of fat, respectively. However, the toxic effects of the dioxins in the milk in this range of concentrations were not explored. Therefore, considering the outbred livestock tissues as better models than inbred laboratory animals, the present study targeted to study the effect of dioxins present in the milk on three-dimensionally (3D) cultured buffalo primary hepatocyte spheroids. The spheroids were treated with a model dioxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), directly and also through milk fat at different concentrations (i.e, 0.02-20 pg/mL) for 24 h. Among the liver-cell-specific (ALB, HNF4α, and AFP) genes, a similar ALB and upregulated HNF4α expression at all treatments indicated the functional and transcriptionally active hepatocyte spheroids. Supportingly, no significant difference in the antiapoptotic gene expression between the treatments of milk fat and milk fat containing dioxins indicated the survivability of the spheroids during dioxin treatments. Among the selected TCDD responsive (CYP1A1, CYP1A2, AHR, CYP1B1, and TIPARP) genes, a nonsignificant increasing trend of the CYP1A1 expression was observed from 0.2 to 10 pg/mL of TCDD concentration through milk fat. This pattern was similar to the reported insensitive response of human primary hepatocytes toward dioxins than that of rat primary hepatocytes. This may indicate that the buffalo hepatocyte spheroids could be better models than rats for TCDD hepatotoxic studies. Further, TCDD in the milk in the range of 0.02-20 pg/mL concentration may not be very hepatotoxic.


Assuntos
Dioxinas/farmacologia , Hepatócitos/efeitos dos fármacos , Leite/química , Animais , Búfalos , Células Cultivadas , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1A2/genética , Citocromo P-450 CYP1A2/metabolismo , Dioxinas/análise , Contaminação de Alimentos/análise , Fator 4 Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/metabolismo , Hepatócitos/metabolismo , Fígado/citologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Modelos Animais , Ratos , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , alfa-Fetoproteínas/genética , alfa-Fetoproteínas/metabolismo
8.
Int J Mol Sci ; 20(12)2019 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-31216773

RESUMO

The Hippo pathway is important for tissue homeostasis, regulation of organ size andgrowth in most tissues. The co-transcription factor yes-associated protein 1 (YAP1) serves as a maindownstream effector of the Hippo pathway and its dysregulation increases cancer development andblocks colonic tissue repair. Nevertheless, little is known about the transcriptional regulation ofYAP1 in intestinal cells. The aim of this study to identify gene control regions in the YAP1 gene andtranscription factors important for intestinal expression. Bioinformatic analysis of caudal typehomeobox 2 (CDX2) and hepatocyte nuclear factor 4 alpha (HNF4α) chromatin immunoprecipitatedDNA from differentiated Caco-2 cells revealed potential intragenic enhancers in the YAP1 gene.Transfection of luciferase-expressing YAP1 promoter-reporter constructs containing the potentialenhancer regions validated one potent enhancer of the YAP1 promoter activity in Caco-2 and T84cells. Two potential CDX2 and one HNF4α binding sites were identified in the enhancer by in silicotranscription factor binding site analysis and protein-DNA binding was confirmed in vitro usingelectrophoretic mobility shift assay. It was found by chromatin immunoprecipitation experimentsthat CDX2 and HNF4α bind to the YAP1 enhancer in Caco-2 cells. These results reveal a previouslyunknown enhancer of the YAP1 promoter activity in the YAP1 gene, with importance for highexpression levels in intestinal epithelial cells. Additionally, CDX2 and HNF4α binding areimportant for the YAP1 enhancer activity in intestinal epithelial cells.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Fator de Transcrição CDX2/metabolismo , Regulação da Expressão Gênica , Fator 4 Nuclear de Hepatócito/metabolismo , Intestinos , Fosfoproteínas/genética , Regiões Promotoras Genéticas , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Cromatina/genética , Cromatina/metabolismo , Elementos Facilitadores Genéticos , Humanos , Ligação Proteica , Fatores de Transcrição
9.
ACS Appl Mater Interfaces ; 11(22): 19808-19818, 2019 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-31066542

RESUMO

Hepatocellular carcinoma (HCC) is one of the most prevalent and deadly malignancies characterized by high rate of recurrence. Tumor recurrence is often attributed to the presence of a subpopulation of cells with stem cell properties, referred to as cancer stem cells (CSCs). Traditionally, cancer therapies target the entire bulk of tumor cells; however, they are poorly effective against CSCs, characterized by higher drug resistance. Therefore, approaches targeting CSCs may be required in addition to conventional chemotherapy to prevent tumor recurrence. In this study, we investigated an approach to target HCC by combining the conventional chemotherapeutic drug, cisplatin, to target the bulk of tumor cells, and differentiation therapy by delivering the gene encoding HNF4α, an important regulator of hepatocyte differentiation, to target CSCs. We used the Huh7 cell line as an in vitro model of HCC, which is characterized by a high proportion of CD133-expressing CSCs. By using flow cytometry, we separated CD133+ and CD133- Huh7 cell subpopulations and have shown that the former has highly pronounced in vivo tumorigenic capacity in contrast to the latter, which could not generate tumors in vivo. For the dual delivery of HNF4α-encoding plasmid and cisplatin, we used polyethyleneimine-modified mesoporous silica nanoparticles (PMSNs) as the nanocarriers. Here, we show that the treatment of CD133-expressing Huh7 cells with HNF4α-loaded PMSNs can suppress their proliferation rate, decrease the proportion of CSCs, downregulate stemness-associated genes, and increase the expression of mature hepatocyte-associated genes. At the same time, the treatment of Huh7 with PMSNs loaded with both HNF4α-encoding plasmid and cisplatin could block them in the S-phase of the cell cycle and cause apoptosis. In addition, dually loaded PMSNs were the most efficient formulation in suppressing tumor growth in vivo. To summarize, in this study, we tested the nanoparticle-based delivery system as both chemotherapy and gene-based therapy agents, which has great potential for development of effective treatment of HCC.


Assuntos
Antígeno AC133/metabolismo , Carcinoma Hepatocelular/metabolismo , Cisplatino/administração & dosagem , Cisplatino/farmacologia , Fator 4 Nuclear de Hepatócito/metabolismo , Nanopartículas/química , Dióxido de Silício/química , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Animais , Western Blotting , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Citometria de Fluxo , Fator 4 Nuclear de Hepatócito/genética , Humanos , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos SCID , Reação em Cadeia da Polimerase Via Transcriptase Reversa
10.
Nat Genet ; 51(5): 777-785, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30988513

RESUMO

BMP/SMAD signaling is a crucial regulator of intestinal differentiation1-4. However, the molecular underpinnings of the BMP pathway in this context are unknown. Here, we characterize the mechanism by which BMP/SMAD signaling drives enterocyte differentiation. We establish that the transcription factor HNF4A acts redundantly with an intestine-restricted HNF4 paralog, HNF4G, to activate enhancer chromatin and upregulate the majority of transcripts enriched in the differentiated epithelium; cells fail to differentiate on double knockout of both HNF4 paralogs. Furthermore, we show that SMAD4 and HNF4 function via a reinforcing feed-forward loop, activating each other's expression and co-binding to regulatory elements of differentiation genes. This feed-forward regulatory module promotes and stabilizes enterocyte cell identity; disruption of the HNF4-SMAD4 module results in loss of enterocyte fate in favor of progenitor and secretory cell lineages. This intersection of signaling and transcriptional control provides a framework to understand regenerative tissue homeostasis, particularly in tissues with inherent cellular plasticity5.


Assuntos
Enterócitos/citologia , Enterócitos/metabolismo , Fator 4 Nuclear de Hepatócito/metabolismo , Proteína Smad4/metabolismo , Animais , Sítios de Ligação/genética , Células CACO-2 , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Elementos Facilitadores Genéticos , Fator 4 Nuclear de Hepatócito/deficiência , Fator 4 Nuclear de Hepatócito/genética , Humanos , Camundongos , Camundongos Knockout , Transdução de Sinais , Proteína Smad4/deficiência , Proteína Smad4/genética
11.
Genome Res ; 29(5): 723-736, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30962179

RESUMO

Esophageal adenocarcinoma (EAC) is one of the most frequent causes of cancer death, and yet compared to other common cancers, we know relatively little about the molecular composition of this tumor type. To further our understanding of this cancer, we have used open chromatin profiling to decipher the transcriptional regulatory networks that are operational in EAC. We have uncovered a transcription factor network that is usually found in primitive intestinal cells during embryonic development, centered on HNF4A and GATA6. These transcription factors work together to control the EAC transcriptome. We show that this network is activated in Barrett's esophagus, the putative precursor state to EAC, thereby providing novel molecular evidence in support of stepwise malignant transition. Furthermore, we show that HNF4A alone is sufficient to drive chromatin opening and activation of a Barrett's-like chromatin signature when expressed in normal human epithelial cells. Collectively, these data provide a new way to categorize EAC at a genome scale and implicate HNF4A activation as a potential pivotal event in its malignant transition from healthy cells.


Assuntos
Adenocarcinoma/genética , Esôfago de Barrett/genética , Neoplasias Esofágicas/genética , Fator de Transcrição GATA6/metabolismo , Redes Reguladoras de Genes/genética , Fator 4 Nuclear de Hepatócito/metabolismo , Adenocarcinoma/metabolismo , Biomarcadores Tumorais/genética , Progressão da Doença , Neoplasias Esofágicas/metabolismo , Feminino , Células HEK293 , Humanos , Masculino , Transcriptoma
12.
Mol Pharmacol ; 95(6): 597-605, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30944208

RESUMO

Overdose of acetaminophen (APAP) is the leading cause of acute liver failure (ALF) in the United States. The sulfotransferase-mediated sulfation of APAP is widely believed to be a protective mechanism to attenuate the hepatotoxicity of APAP. The cholesterol sulfotransferase SULT2B1b is best known for its activity in catalyzing the sulfoconjugation of cholesterol to synthesize cholesterol sulfate. SULT2B1b can be transcriptionally and positively regulated by the hepatic nuclear factor 4α (HNF4α). In this study, we uncovered an unexpected role for SULT2B1b in APAP toxicity. Hepatic overexpression of SULT2B1b sensitized mice to APAP-induced liver injury, whereas ablation of the Sult2B1b gene in mice conferred resistance to the APAP hepatotoxicity. Consistent with the notion that Sult2B1b is a transcriptional target of HNF4α, overexpression of HNF4α sensitized mice or primary hepatocytes to APAP-induced hepatotoxicity in a Sult2B1b-dependent manner. We conclude that the HNF4α-SULT2B1b axis has a unique role in APAP-induced acute liver injury, and SULT2B1b induction might be a risk factor for APAP hepatotoxicity.


Assuntos
Acetaminofen/efeitos adversos , Doença Hepática Crônica Induzida por Substâncias e Drogas/metabolismo , Overdose de Drogas/complicações , Fator 4 Nuclear de Hepatócito/metabolismo , Sulfotransferases/genética , Animais , Células Cultivadas , Doença Hepática Crônica Induzida por Substâncias e Drogas/genética , Modelos Animais de Doenças , Overdose de Drogas/etiologia , Overdose de Drogas/metabolismo , Feminino , Hepatócitos/citologia , Hepatócitos/metabolismo , Camundongos , Sulfotransferases/metabolismo
13.
Lipids ; 54(1): 39-51, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30740707

RESUMO

In this study, the effect of n-3 fatty acids (FA) [α-linolenic acid (ALA) and eicosapentaenoic acid (EPA) + docosahexaenoic acid (DHA)] on the intestinal bile acid (BA) uptake, hepatic BA synthesis, and enterohepatic bile acid transporters (BAT) was assessed in young and aged dyslipidemic rats. Dyslipidemia was induced in young and aged rats by feeding a high-fat (HF) diet. Experimental groups received diets containing canola oil (HF + CNO) and fish oil (HF + FO) as a source of ALA and EPA + DHA, respectively. After 60 days of feeding, intestinal BA uptake and expression of apical sodium-dependent bile acid transporter (Asbt), organic solute transporter-alpha/beta (Osta/b) messenger RNA (mRNA), and hepatic expression of Na+ taurocholate cotransporting polypeptide (Ntcp), bile salt export pump (Bsep), cholesterol 7-α hydroxylase A1 (Cyp7a1), Farnesoid X receptor (Fxr), small heterodimer partner-1 (Shp), liver receptor homolog-1 (Lrh-1), and hepatic nuclear factor-4 alpha (Hnf4a) mRNA were measured. Hepatic 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase activity and total BA in serum, liver, and feces were assessed. The dyslipidemic HF group had: (1) increased intestinal BA uptake and Asbt and Osta/b mRNA expression, (2) increased BA in serum, (3) decreased hepatic expression of Ntcp, Bsep, and Cyp7a1 mRNA, (4) increased activity of HMG-CoA reductase, (5) increased hepatic expression of Fxr and Shp mRNA, (6) decreased hepatic expression of Lrh-1 and Hnf4a mRNA, and (7) decreased BA in feces, when compared to control, HF + CNO, and HF + FO groups. Immunostaining revealed increased expression of intestinal Asbt and hepatic Ntcp protein in the HF group when compared to control, HF + CNO, and HF + FO groups. n-3 FA abrogated dyslipidemia-induced changes in the intestinal uptake, hepatic synthesis, and enterohepatic transporters of BA in both young and aged rats. EPA + DHA was more effective than ALA in modulating dyslipidemia-induced changes.


Assuntos
Ácidos e Sais Biliares/metabolismo , Proteínas de Transporte/metabolismo , Dislipidemias/tratamento farmacológico , Dislipidemias/metabolismo , Ácidos Graxos Ômega-3/uso terapêutico , Glicoproteínas de Membrana/metabolismo , Animais , Ácidos e Sais Biliares/sangue , Colesterol 7-alfa-Hidroxilase/metabolismo , Ácidos Docosa-Hexaenoicos/uso terapêutico , Dislipidemias/sangue , Ácido Eicosapentaenoico/uso terapêutico , Fezes/química , Fator 4 Nuclear de Hepatócito/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Ratos , Ratos Wistar , Ácido alfa-Linoleico/uso terapêutico
14.
Cell Biol Int ; 43(5): 476-485, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30761668

RESUMO

There are numerous studies which provide support for the use of human adipose tissue-derived stem cells (hASCs) to generate hepatocyte-like cells. However, the produced cells exhibit only a certain level of differentiation, mainly due to inefficient induction conditions. Therefore, based on the important role of insulin-like growth factor (IGF-I) in hepatic function and development, in the current study we evaluated the differentiation efficacy of the mentioned factor to induce hASCs into functional hepatocyte-like cells. To investigate this, using a two-step protocol, hASCs were treated with a combination of HGF, Dex, and OSM in the presence or absence of IGF-I up to 21 days. Hepatic differentiation was evaluated by analyzing specific hepatocyte markers at different time points of differentiation induction. Increased expression of hepatocyte-specific genes including ALB, AFP, CK18, and HNF4a, downregulation of bile duct cells marker (CK19), the higher number of ALB positive cells, increased urea production together with higher glycogen deposit was observed upon the treatment of hASCs with the induction medium containing IGF-I compared to the other treatment. In conclusion, our findings suggest IGF-I as a potent inducer of hepatic differentiation of hASCs and its potential to generate more functional hepatocyte-like cells.


Assuntos
Técnicas de Cultura de Células/métodos , Hepatócitos/metabolismo , Fator de Crescimento Insulin-Like I/fisiologia , Tecido Adiposo/citologia , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Células Cultivadas , Fator de Crescimento de Hepatócito/metabolismo , Fator 4 Nuclear de Hepatócito/metabolismo , Hepatócitos/citologia , Humanos , Fator de Crescimento Insulin-Like I/metabolismo , Queratina-18/metabolismo , Fígado/metabolismo , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/fisiologia , Albumina Sérica Humana/metabolismo , Células-Tronco/citologia , alfa-Fetoproteínas/metabolismo
15.
Mutat Res ; 814: 1-6, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30648609

RESUMO

HNF4α is a culprit gene product for a monogenic and dominantly-inherited form of diabetes, referred to as MODY1 (Maturity Onset Diabetes of the Young type 1). Reduced HNF4α activities have been linked to impaired insulin secretion and ß-cell function. Numerous mutations have been identified from the patients and they have been instructive as to the individual residue's role in protein structure-function and dysfunction. As a member of the nuclear receptor (NR) superfamily, HNF4α is made of characteristic modular domains and it functions exclusively as a homodimer despite its sequence homology to RXR, a common heterodimer partner of non-steroidal NRs. Transcription factors commonly dimerize to enhance their molecular functions mainly by facilitating the recognition of double helix target DNAs that display an intrinsic pseudo-2-fold symmetry and the recruitment of the remainder of the main transcriptional machinery. HNF4α is no exception and its dimerization is maintained by the ligand binding domain (LBD) mainly through the leucine-zipper-like interactions at the stalk of two interacting helices. Although many MODY1 mutations have been previously characterized, including DNA binding disruptors, ligand binding disruptors, coactivator binding disruptors, and protein stability disruptors, protein dimerization disruptors have not been formally reported. In this report, we present a set of data for the two MODY1 mutations found right at the dimerization interface (L332 P and L328del mutations) which clearly exhibit the disruptive effects of directly affecting dimerization, protein stability, and transcriptional activities. These data reinforced the fact that MODY mutations are loss-of-function mutations and HNF4α dimerization is essential for its optimal function and normal physiology.


Assuntos
Diabetes Mellitus Tipo 2/genética , Fator 4 Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/metabolismo , Mutação , Domínios e Motivos de Interação entre Proteínas/genética , Multimerização Proteica , Dimerização , Células HeLa , Fator 4 Nuclear de Hepatócito/química , Humanos , Mutação com Perda de Função/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Ligação Proteica , Multimerização Proteica/genética , Estabilidade Proteica , Estrutura Quaternária de Proteína/genética , Ativação Transcricional/genética
16.
Int J Mol Sci ; 20(2)2019 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-30654452

RESUMO

Although human liver tumor cells have reduced metabolic functions as compared to primary human hepatocytes (PHH) they are widely used for pre-screening tests of drug metabolism and toxicity. The aim of the present study was to modify liver cancer cell lines in order to improve their drug-metabolizing activities towards PHH. It is well-known that epigenetics is strongly modified in tumor cells and that epigenetic regulators influence the expression and function of Cytochrome P450 (CYP) enzymes through altering crucial transcription factors responsible for drug-metabolizing enzymes. Therefore, we screened the epigenetic status of four different liver cancer cell lines (Huh7, HLE, HepG2 and AKN-1) which were reported to have metabolizing drug activities. Our results showed that HepG2 cells demonstrated the highest similarity compared to PHH. Thus, we modified the epigenetic status of HepG2 cells towards 'normal' liver cells by 5-Azacytidine (5-AZA) and Vitamin C exposure. Then, mRNA expression of Epithelial-mesenchymal transition (EMT) marker SNAIL and CYP enzymes were measured by PCR and determinate specific drug metabolites, associated with CYP enzymes by LC/MS. Our results demonstrated an epigenetic shift in HepG2 cells towards PHH after exposure to 5-AZA and Vitamin C which resulted in a higher expression and activity of specific drug metabolizing CYP enzymes. Finally, we observed that 5-AZA and Vitamin C led to an increased expression of Hepatocyte nuclear factor 4α (HNF4α) and E-Cadherin and a significant down regulation of Snail1 (SNAIL), the key transcriptional repressor of E-Cadherin. Our study shows, that certain phase I genes and their enzyme activities are increased by epigenetic modification in HepG2 cells with a concomitant reduction of EMT marker gene SNAIL. The enhancing of liver specific functions in hepatoma cells using epigenetic modifiers opens new opportunities for the usage of cell lines as a potential liver in vitro model for drug testing and development.


Assuntos
Epigênese Genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Preparações Farmacêuticas/metabolismo , Ácido Ascórbico/farmacologia , Azacitidina/farmacologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Cromatina/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Regulação para Baixo/efeitos dos fármacos , Epigênese Genética/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células Hep G2 , Fator 4 Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Hidrocortisona/farmacologia , Insulina/farmacologia , Neoplasias Hepáticas/enzimologia , Fatores de Transcrição da Família Snail/metabolismo , Xenobióticos/metabolismo
17.
Mol Imaging Biol ; 21(1): 44-53, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-29761416

RESUMO

PURPOSE: The purposes of this study were to construct immortalized human bone marrow mesenchymal stem cells (UE7T-13) with overexpression of the hepatocyte nuclear factor4α (hHNF4α) and luciferase2-mKate2 dual-fusion reporter gene, further investigate their impact on treating acute liver injury (ALI) in rats, and track their biodistribution and survival by bioluminescence imaging (BLI). PROCEDURES: The hHNF4α and luciferase2-mKate2 genes were transduced by a lentiviral vector into UE7T-13 cells (named E7-hHNF4α-R cells), and expression was verified by immunofluorescence, RT-PCR, and flow cytometry. E7-hGFP-R cells expressing the luciferase2-mKate2/hGFP gene served as a negative group. A correlation between the bioluminescence signal and cell number was detected by BLI. The ALI rats were established and divided into three groups: PBS, E7-hGFP-R, and E7-hHNF4α-R. After transplantation of 2.0 × 106 cells, BLI was used to dynamically track their biodistribution and survival. The restoration of biological functions was assessed by serum biochemical and histological analyses. RESULTS: Stable high-level expression of hHNF4α and mKate2 protein was established in the E7-hHNF4α-R cells in vitro. The E7-hHNF4α-R cells strongly expressed hGFP, hHNF4α, and mKate2 proteins, and the hHNF4α gene. hGFP-mKate2 dual-positive cell expression reached approximately 93 %. BLI verified that a linear relationship existed between the cell number and bioluminescence signal (R2 = 0.9991). The cells improved liver function in vivo after transplantation into the ALI rat liver, as evidenced by the fact that AST and ALT temporarily returned to normal levels in the recipient ALI rats. The presence of the transplanted E7-hGFP-R and E7-hHNF4α-R cells in recipient rat livers was confirmed by BLI and immunohistochemistry. However, the cells were cleared by the immune system a short time after transplantation into ALI rats with a normal immune system. CONCLUSION: Our data revealed that the E7-hHNF4α-R cells can transiently improve damaged liver function and were rapidly cleared by the immune system. In addition, BLI is a useful tool to track transplanted cell biodistribution and survival.


Assuntos
Rastreamento de Células , Fator 4 Nuclear de Hepatócito/metabolismo , Medições Luminescentes/métodos , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Animais , Sobrevivência Celular , Feminino , Humanos , Lentivirus/genética , Fígado/lesões , Fígado/metabolismo , Fígado/patologia , Luciferases/metabolismo , Ratos Sprague-Dawley , Distribuição Tecidual
18.
J Steroid Biochem Mol Biol ; 187: 118-123, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30468856

RESUMO

In higher metazoans, the nuclear hormone receptors activate transcription trough their specific adaptors, nuclear hormone receptor adaptors NCoA, which are absent in lower metazoans. The Nine amino acid TransActivation Domain, 9aaTAD, was reported for a large number of the transcription activators that recruit general mediators of transcription. In this study, we demonstrated that the 9aaTAD from NHR-49 receptor of nematode C.elegans activates transcription as a small peptide. We showed that the ancient 9aaTAD domains are conserved in the nuclear hormone receptors including human HNF4, RARa, VDR and PPARg. Also their small 9aaTAD peptides effectively activated transcription in absence of the NCoA adaptors. We also showed that adjacent H11 domains in ancient and modern hormone receptors have an inhibitory effect on their 9aaTAD function.


Assuntos
Receptores Citoplasmáticos e Nucleares/metabolismo , Ativação Transcricional , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/química , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/metabolismo , Fator 4 Nuclear de Hepatócito/química , Fator 4 Nuclear de Hepatócito/metabolismo , Humanos , Modelos Moleculares , PPAR gama/química , PPAR gama/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Domínios Proteicos , Receptores de Calcitriol/química , Receptores de Calcitriol/metabolismo , Receptores Citoplasmáticos e Nucleares/química , Receptor alfa de Ácido Retinoico/química , Receptor alfa de Ácido Retinoico/metabolismo , Alinhamento de Sequência , Transdução de Sinais
19.
Int J Mol Med ; 43(2): 1021-1032, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30535491

RESUMO

Hepatocyte nuclear factor 4 α (HNF­4α) is a nuclear receptor and mediates hepatic genes. WB­F344 liver epithelial cells can differentiate into hepatocytes. The present study aimed to examine the roles and mechanisms of action of HNF­4α on the hepatic differentiation of WB­F344 cells. WB­F344 cells were divided into a normal cell group (WB­F344), empty vector group (PLKO), and gene silencing group (PLKO­SH). The expression levels of HNF­4α were measured using reverse transcription­quantitative polymerase chain reaction analysis. Proliferation of the cells was determined using a Cell Counting kit­8 assay. Based on western blot analysis, the protein levels of α­fetoprotein (AFP), albumin (ALB) and cytokeratin 19 (CK19) were determined. The positive cell rates of the three groups were assessed using periodic acid­Schiff (PAS) staining. Following construction of an RNA­sequencing library, differentially expressed genes (DEGs) between the HNF­4α­silenced and normal samples were screened using the limma package and enrichment analysis was conducted using the DAVID tool. Protein­protein interaction (PPI) and microRNA­targeted regulatory networks were constructed in Cytoscape software. The PLKO­SH group exhibited a lower mRNA level of HNF­4α, higher protein level of AFP, lower protein levels of ALB and CK19, increased cell proliferation, and a lower PAS­positive cell rate. The HNF­4α­silenced and normal samples differed in 499 DEGs. In the PPI network, matrix metallopeptidase 9 (MMP9), early growth response 1 (EGR1), SMAD family member 2 (SMAD2), and RAS­related C3 botulinum substrate 2 (RAC2) were key nodes. HNF­4α may promote the differentiation of WB­F344 cells into hepatocytes by targeting MMP9, EGR1, SMAD2 and RAC2.


Assuntos
Diferenciação Celular/genética , Fator 4 Nuclear de Hepatócito/genética , Hepatócitos/metabolismo , Linhagem Celular Tumoral , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Fator 4 Nuclear de Hepatócito/metabolismo , Humanos , MicroRNAs/genética , Mapeamento de Interação de Proteínas , Mapas de Interação de Proteínas , RNA Mensageiro/genética
20.
Methods Mol Biol ; 1905: 29-41, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30536088

RESUMO

This protocol showed procedures to isolate and expand small hepatocytes (SHs), as hepatocytic progenitor cells, from a rat liver. SHs are identified as a subpopulation of mature hepatocytes in a healthy liver. SHs can proliferate to form colonies in serum-free medium on hyaluronic acid-coated dishes, of which cells show CD44 positivity (CD44+ SHs). CD44+ SHs can be separated and purified from colonies by using anti-CD44 antibodies after enzymatic dissociation. CD44+ SHs can proliferate to form colonies on Engelbreth-Holm-Swarm gel (EHS-gel)-coated dishes in the serum-free medium for a long period and subculture for several times. Even after the second passage, the cells possess characteristics of hepatocytes such as expression of albumin and HNF4α. In addition, when the cells are treated with EHS-gel, they can recover highly differentiated functions of hepatocytes such as glycogen production, CYP activity, and bile secretion.


Assuntos
Técnicas de Cultura de Células/métodos , Separação Celular/métodos , Hepatócitos/citologia , Células-Tronco/citologia , Albuminas/metabolismo , Animais , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Meios de Cultura Livres de Soro/química , Fator 4 Nuclear de Hepatócito/metabolismo , Hepatócitos/metabolismo , Receptores de Hialuronatos/metabolismo , Ratos , Células-Tronco/metabolismo
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