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1.
Cell Prolif ; 54(2): e12978, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33393114

RESUMO

OBJECTIVES: The final stage of liver development is the production of hepatocytes and cholangiocytes (biliary epithelial cells) from bipotent hepatic progenitor cells. We used HepaRG cells, which are bipotent and able to differentiate into both hepatocytes and cholangiocytes, as a model to study the action of a novel lncRNA (lnc-RHL) and its role in the regulation of bipotency leading to hepatocytes and cholangiocytes. MATERIALS AND METHODS: Differentiation of HepaRG cells was assessed by marker expression and morphology which revealed their ability to differentiate into hepatocytes and cholangiocytes (modelling the behaviour of hepatoblasts in vivo). Using a qRT-PCR and RACE, we cloned a novel lncRNA (lnc-RHL; regulator of hepatic lineages) that is upregulated upon HepaRG differentiation. Using inducible knockdown of lnc-RHL concurrently with differentiation, we show that lnc-RHL is required for proper HepaRG cell differentiation resulting in diminution of the hepatocyte lineage. RESULTS: Here, we report the discovery of lnc-RHL, a spliced and polyadenylated 670 base lncRNA expressed from the 11q23.3 apolipoprotein gene cluster. lnc-RHL expression is confined to hepatic lineages and is upregulated when bipotent HepaRG cells are caused to differentiate. HepaRG cells made deficient for lnc-RHL have reduced ability to differentiate into hepatocytes, but retain their ability to differentiate into cholangiocytes. CONCLUSIONS: Deficiency for lnc-RHL in HepaRG cells converts them from bipotent progenitor cells to unipotent progenitor cells with impaired ability to yield hepatocytes. We conclude that lnc-RHL is a key regulator of bipotency in HepaRG cells.


Assuntos
Diferenciação Celular/genética , Hepatócitos/metabolismo , RNA Longo não Codificante/metabolismo , Apolipoproteínas/genética , Apolipoproteínas/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem da Célula , Criança , Cromossomos Humanos Par 11 , Doxorrubicina/farmacologia , Feminino , Células Hep G2 , Fator 4 Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/metabolismo , Hepatócitos/citologia , Humanos , Fígado/metabolismo , Masculino , Família Multigênica , Interferência de RNA , RNA Longo não Codificante/antagonistas & inibidores , RNA Longo não Codificante/genética , RNA Interferente Pequeno/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Adulto Jovem
2.
Am J Chin Med ; 48(6): 1435-1454, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32907363

RESUMO

Endoplasmic reticulum stress (ER stress) plays a main role in pancreatic [Formula: see text]-cell dysfunction and death because of intracellular Ca[Formula: see text] turbulence and inflammation activation. Although several drugs are targeting pancreatic [Formula: see text]-cell to improve [Formula: see text]-cell function, there still lacks agents to alleviate [Formula: see text]-cell ER stress conditions. Therefore we used thapsigargin (THAP) or high glucose (HG) to induce ER stress in [Formula: see text]-cell and aimed to screen natural molecules against ER stress-induced [Formula: see text]-cell dysfunction. Through screening the Traditional Chinese drug library ([Formula: see text] molecules), luteolin was finally discovered to improve [Formula: see text]-cell function. Cellular viability results indicated luteolin reduced the THAP or HG-induced [Formula: see text]-cell death and apoptosis through MTT and flow cytometry assay. Moreover, luteolin improved [Formula: see text]-cell insulin secretion ability under ER stress conditions. Also ER stress-induced intracellular Ca[Formula: see text] turbulence and inflammation activation were inhibited by luteolin treatment. Mechanically, luteolin inhibited HNF4[Formula: see text] signaling, which was induced by ER stress. Moreover, luteolin reduced the transcriptional level of HNF4[Formula: see text] downstream gene, such as Asnk4b and HNF1[Formula: see text]. Conversely HNF4[Formula: see text] knockdown abolished the effect of luteolin on [Formula: see text]-cell using siRNA. These results suggested the protective effect of luteolin on [Formula: see text]-cell was through HNF4[Formula: see text]/Asnk4b pathway. In conclusion, our study discovered that luteolin improved [Formula: see text]-cell function and disclosed the underlying mechanism of luteolin on [Formula: see text]-cell, suggesting luteolin is a promising agent against pancreatic dysfunction.


Assuntos
Sobrevivência Celular/efeitos dos fármacos , Medicamentos de Ervas Chinesas/química , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/genética , Fator 4 Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/metabolismo , Células Secretoras de Insulina/patologia , Células Secretoras de Insulina/fisiologia , Luteolina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Tapsigargina/efeitos adversos , Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Morte Celular/efeitos dos fármacos , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos , Estresse do Retículo Endoplasmático/fisiologia , Glucose/efeitos adversos , Células Secretoras de Insulina/metabolismo , Luteolina/isolamento & purificação
3.
Mol Cell ; 79(4): 660-676.e8, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32755593

RESUMO

Specific combinations of two transcription factors (Hnf4α plus Foxa1, Foxa2, or Foxa3) can induce direct conversion of mouse fibroblasts into hepatocyte-like cells. However, the molecular mechanisms underlying hepatic reprogramming are largely unknown. Here, we show that the Foxa protein family members and Hnf4α sequentially and cooperatively bind to chromatin to activate liver-specific gene expression. Although all Foxa proteins bind to and open regions of closed chromatin as pioneer factors, Foxa3 has the unique potential of transferring from the distal to proximal regions of the transcription start site of target genes, binding RNA polymerase II, and co-traversing target genes. These distinctive characteristics of Foxa3 are essential for inducing the hepatic fate in fibroblasts. Similar functional coupling of transcription factors to RNA polymerase II may occur in other contexts whereby transcriptional activation can induce cell differentiation.


Assuntos
Fator 3-gama Nuclear de Hepatócito/metabolismo , Fator 4 Nuclear de Hepatócito/metabolismo , Fígado/citologia , Fígado/fisiologia , Ativação Transcricional , Animais , Sítios de Ligação , Células Cultivadas , Reprogramação Celular/fisiologia , Cromatina/metabolismo , DNA Polimerase II/genética , DNA Polimerase II/metabolismo , Fibroblastos/citologia , Fibroblastos/fisiologia , Regulação da Expressão Gênica , Fator 3-gama Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/genética , Camundongos Endogâmicos C57BL , Domínios Proteicos , Sítio de Iniciação de Transcrição
4.
Nat Commun ; 11(1): 3978, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32770044

RESUMO

Methionine restriction, a dietary regimen that protects against metabolic diseases and aging, represses cancer growth and improves cancer therapy. However, the response of different cancer cells to this nutritional manipulation is highly variable, and the molecular determinants of this heterogeneity remain poorly understood. Here we report that hepatocyte nuclear factor 4α (HNF4α) dictates the sensitivity of liver cancer to methionine restriction. We show that hepatic sulfur amino acid (SAA) metabolism is under transcriptional control of HNF4α. Knocking down HNF4α or SAA enzymes in HNF4α-positive epithelial liver cancer lines impairs SAA metabolism, increases resistance to methionine restriction or sorafenib, promotes epithelial-mesenchymal transition, and induces cell migration. Conversely, genetic or metabolic restoration of the transsulfuration pathway in SAA metabolism significantly alleviates the outcomes induced by HNF4α deficiency in liver cancer cells. Our study identifies HNF4α as a regulator of hepatic SAA metabolism that regulates the sensitivity of liver cancer to methionine restriction.


Assuntos
Fator 4 Nuclear de Hepatócito/metabolismo , Neoplasias Hepáticas/metabolismo , Metionina/metabolismo , Animais , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Movimento Celular/genética , Cisteína/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Fator 4 Nuclear de Hepatócito/genética , Fígado/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Mesoderma/efeitos dos fármacos , Mesoderma/patologia , Redes e Vias Metabólicas/efeitos dos fármacos , Metaboloma/efeitos dos fármacos , Camundongos , Sorafenibe/farmacologia , Transcrição Genética/efeitos dos fármacos
5.
Proc Natl Acad Sci U S A ; 117(29): 17177-17186, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32631996

RESUMO

Hepatocyte nuclear factor 4α (HNF4α) is a master regulator of liver function and a tumor suppressor in hepatocellular carcinoma (HCC). In this study, we explore the reciprocal negative regulation of HNF4α and cyclin D1, a key cell cycle protein in the liver. Transcriptomic analysis of cultured hepatocyte and HCC cells found that cyclin D1 knockdown induced the expression of a large network of HNF4α-regulated genes. Chromatin immunoprecipitation-sequencing (ChIP-seq) demonstrated that cyclin D1 inhibits the binding of HNF4α to thousands of targets in the liver, thereby diminishing the expression of associated genes that regulate diverse metabolic activities. Conversely, acute HNF4α deletion in the liver induces cyclin D1 and hepatocyte cell cycle progression; concurrent cyclin D1 ablation blocked this proliferation, suggesting that HNF4α maintains proliferative quiescence in the liver, at least, in part, via repression of cyclin D1. Acute cyclin D1 deletion in the regenerating liver markedly inhibited hepatocyte proliferation after partial hepatectomy, confirming its pivotal role in cell cycle progression in this in vivo model, and enhanced the expression of HNF4α target proteins. Hepatocyte cyclin D1 gene ablation caused markedly increased postprandial liver glycogen levels (in a HNF4α-dependent fashion), indicating that the cyclin D1-HNF4α axis regulates glucose metabolism in response to feeding. In AML12 hepatocytes, cyclin D1 depletion led to increased glucose uptake, which was negated if HNF4α was depleted simultaneously, and markedly elevated glycogen synthesis. To summarize, mutual repression by cyclin D1 and HNF4α coordinately controls the cell cycle machinery and metabolism in the liver.


Assuntos
Ciclo Celular/fisiologia , Ciclina D1/genética , Ciclina D1/metabolismo , Fator 4 Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/metabolismo , Fígado/metabolismo , Animais , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Modelos Animais de Doenças , Feminino , Técnicas de Silenciamento de Genes , Hepatócitos/metabolismo , Hepatócitos/patologia , Regeneração Hepática/genética , Regeneração Hepática/fisiologia , Masculino , Camundongos Endogâmicos BALB C , Camundongos Knockout
6.
Prostate ; 80(12): 962-976, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32511787

RESUMO

OBJECTIVE: The broad goal of the research described in this study was to investigate the contributions of selenium-binding protein 1 (SBP1) loss in prostate cancer development and outcome. METHODS: SBP1 levels were altered in prostate cancer cell lines and the consequences on oxygen consumption, expression of proteins associated with energy metabolism, and cellular transformation and migration were investigated. The effects of exposing cells to the SBP1 reaction products, H2 O2 and H2 S were also assessed. In silico analyses identified potential HNF4α binding sites within the SBP1 promoter region and this was investigated using an inhibitor specific for that transcription factor. RESULTS: Using in silico analyses, it was determined that the promoter region of SBP1 contains putative binding sites for the HNF4α transcription factor. The potential for HNF4α to regulate SBP1 expression was supported by data indicating that HNF4α inhibition resulted in a dose-response increase in the levels of SBP1 messenger RNA and protein, identifying HNF4α as a novel negative regulator of SBP1 expression in prostate cancer cells. The consequences of altering the levels of SBP1 were investigated by ectopically expressing SBP1 in PC-3 prostate cancer cells, where SBP1 expression attenuated anchorage-independent cellular growth and migration in culture, both properties associated with transformation. SBP1 overexpression reduced oxygen consumption in these cells and increased the activation of AMP-activated protein kinase (AMPK), a major regulator of energy homeostasis. In addition, the reaction products of SBP1, H2 O2 , and H2 S also activated AMPK. CONCLUSIONS: Based on the obtained data, it is hypothesized that SBP1 negatively regulates oxidative phosphorylation (OXPHOS) in the healthy prostate cells by the production of H2 O2 and H2 S and consequential activation of AMPK. The reduction of SBP1 levels in prostate cancer can occur due to increased binding of HNF4α, acting as a transcriptional inhibitor to the SBP1 promoter. Consequently, there is a reduction in H2 O2 and H2 S-mediated signaling, inhibition of AMPK, and stimulation of OXPHOS and building blocks of biomolecules needed for tumor growth and progression. Other effects of SBP1 loss in tumor cells remain to be discovered.


Assuntos
Neoplasias da Próstata/metabolismo , Proteínas de Ligação a Selênio/metabolismo , Linhagem Celular Tumoral , Transformação Celular Viral , Metilação de DNA , Progressão da Doença , Metabolismo Energético , Regulação Neoplásica da Expressão Gênica , Glucose/metabolismo , Fator 4 Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Sulfeto de Hidrogênio/metabolismo , Masculino , Fosforilação Oxidativa , Consumo de Oxigênio , Células PC-3 , Regiões Promotoras Genéticas , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Proteínas Quinases/metabolismo , Proteínas de Ligação a Selênio/deficiência , Proteínas de Ligação a Selênio/genética , Frações Subcelulares/metabolismo
7.
Sci Rep ; 10(1): 7822, 2020 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-32385352

RESUMO

A basic question linked to differential patterns of gene expression is how cells reach different fates despite using the same DNA template. Since 5-hydroxymethylcytosine (5hmC) emerged as an intermediate metabolite in active DNA demethylation, there have been increasing efforts to elucidate its function as a stable modification of the genome, including a role in establishing such tissue-specific patterns of expression. Recently we described TET1-mediated enrichment of 5hmC on the promoter region of the master regulator of hepatocyte identity, HNF4A, which precedes differentiation of liver adult progenitor cells in vitro. Here, we studied the genome-wide distribution of 5hmC at early in vitro differentiation of human hepatocyte-like cells. We found a global increase in 5hmC as well as a drop in 5-methylcytosine after one week of in vitro differentiation from bipotent progenitors, at a time when the liver transcript program is already established. 5hmC was overall higher at the bodies of overexpressed genes. Furthermore, by modifying the metabolic environment, an adenosine derivative prevents 5hmC enrichment and impairs the acquisition of hepatic identity markers. These results suggest that 5hmC could be a marker of cell identity, as well as a useful biomarker in conditions associated with cell de-differentiation such as liver malignancies.


Assuntos
5-Metilcitosina/análogos & derivados , Diferenciação Celular/genética , Metilação de DNA/genética , Fator 4 Nuclear de Hepatócito/genética , Oxigenases de Função Mista/genética , Proteínas Proto-Oncogênicas/genética , 5-Metilcitosina/metabolismo , Desmetilação do DNA , Regulação da Expressão Gênica no Desenvolvimento/genética , Genoma/genética , Hepatócitos/metabolismo , Humanos , Regiões Promotoras Genéticas/genética , Células-Tronco/metabolismo
8.
Am J Physiol Cell Physiol ; 318(6): C1294-C1304, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32348179

RESUMO

The serotonin transporter (SERT) functions to regulate the availability of serotonin (5-HT) in the brain and intestine. An intestine-specific mRNA variant arising from a unique transcription start site and alternative promoter in the SERT gene has been identified (iSERT; spanning exon 1C). A decrease in SERT is implicated in several gut disorders, including inflammatory bowel diseases (IBD). However, little is known about mechanisms regulating the iSERT variant, and a clearer understanding is warranted for targeting SERT for the treatment of gut disorders. The current studies examined the expression of iSERT across different human intestinal regions and investigated its regulation by HNF4α (hepatic nuclear factor-4α), a transcription factor important for diverse cellular functions. iSERT mRNA abundance was highest in the human ileum and Caco-2 cell line. iSERT mRNA expression was downregulated by loss of HNF4α (but not HNF1α, HNF1ß, or FOXA1) in Caco-2 cells. Overexpression of HNF4α increased iSERT mRNA concomitant with an increase in SERT protein. Progressive promoter deletion and site-directed mutagenesis revealed that the HNF4α response element spans nucleotides -1,163 to -1150 relative to the translation start site. SERT mRNA levels in the intestine were drastically reduced in the intestine-specific HNF4α-knockout mice relative to HNF4αFL/FL mice. Both HNF4α and SERT mRNA levels were also downregulated in mouse model of ileitis (SAMP) compared with AKR control mice. These results establish the transcriptional regulation of iSERT at the gut-specific internal promoter (hSERTp2) and have identified HNF4α as a critical modulator of basal SERT expression in the intestine.


Assuntos
Células Epiteliais/metabolismo , Fator 4 Nuclear de Hepatócito/metabolismo , Ileíte/metabolismo , Íleo/metabolismo , Mucosa Intestinal/metabolismo , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Animais , Células CACO-2 , Modelos Animais de Doenças , Células Epiteliais/patologia , Fator 4 Nuclear de Hepatócito/deficiência , Fator 4 Nuclear de Hepatócito/genética , Humanos , Ileíte/genética , Ileíte/patologia , Íleo/patologia , Mucosa Intestinal/patologia , Masculino , Camundongos Knockout , Regiões Promotoras Genéticas , Elementos de Resposta , Proteínas da Membrana Plasmática de Transporte de Serotonina/genética , Transcrição Genética
9.
Diabetes ; 69(6): 1126-1139, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32312868

RESUMO

Mitochondrial protein FAM3A suppresses hepatic gluconeogenesis and lipogenesis. This study aimed to screen drug(s) that activates FAM3A expression and evaluate its effect(s) on hyperglycemia and steatosis. Drug-repurposing methodology predicted that antidepressive drug doxepin was among the drugs that potentially activated FAM3A expression. Doxepin was further validated to stimulate the translocation of transcription factor HNF4α from the cytoplasm into the nucleus, where it promoted FAM3A transcription to enhance ATP synthesis, suppress gluconeogenesis, and reduce lipid deposition in hepatocytes. HNF4α antagonism or FAM3A deficiency blunted doxepin-induced suppression on gluconeogenesis and lipid deposition in hepatocytes. Doxepin administration attenuated hyperglycemia, steatosis, and obesity in obese diabetic mice with upregulated FAM3A expression in liver and brown adipose tissues (BAT). Notably, doxepin failed to correct dysregulated glucose and lipid metabolism in FAM3A-deficient mice fed on high-fat diet. Doxepin's effects on ATP production, Akt activation, gluconeogenesis, and lipogenesis repression were also blunted in FAM3A-deficient mouse livers. In conclusion, FAM3A is a therapeutic target for diabetes and steatosis. Antidepressive drug doxepin activates FAM3A signaling pathways in liver and BAT to improve hyperglycemia and steatosis of obese diabetic mice. Doxepin might be preferentially recommended as an antidepressive drug in potential treatment of patients with diabetes complicated with depression.


Assuntos
Citocinas/metabolismo , Doxepina/uso terapêutico , Reposicionamento de Medicamentos , Fígado Gorduroso/tratamento farmacológico , Regulação da Expressão Gênica/efeitos dos fármacos , Hiperglicemia/tratamento farmacológico , Animais , Antidepressivos Tricíclicos/uso terapêutico , Citocinas/genética , Bases de Dados de Produtos Farmacêuticos , Proteína Forkhead Box O1/metabolismo , Células Hep G2 , Fator 4 Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/metabolismo , Hepatócitos/metabolismo , Humanos , Masculino , Camundongos , Camundongos Knockout
10.
Cancer Res ; 80(13): 2722-2736, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32332020

RESUMO

Gastrointestinal adenocarcinomas (GIAC) of the tubular gastrointestinal (GI) tract including esophagus, stomach, colon, and rectum comprise most GI cancers and share a spectrum of genomic features. However, the unified epigenomic changes specific to GIAC are poorly characterized. Using 907 GIAC samples from The Cancer Genome Atlas, we applied mathematical algorithms to large-scale DNA methylome and transcriptome profiles to reconstruct transcription factor (TF) networks and identify a list of functionally hyperactive master regulator (MR) TF shared across different GIAC. The top candidate HNF4A exhibited prominent genomic and epigenomic activation in a GIAC-specific manner. A complex interplay between the HNF4A promoter and three distal enhancer elements was coordinated by GIAC-specific MRTF including ELF3, GATA4, GATA6, and KLF5. HNF4A also self-regulated its own promoter and enhancers. Functionally, HNF4A promoted cancer proliferation and survival by transcriptional activation of many downstream targets, including HNF1A and factors of interleukin signaling, in a lineage-specific manner. Overall, our study provides new insights into the GIAC-specific gene regulatory networks and identifies potential therapeutic strategies against these common cancers. SIGNIFICANCE: These findings show that GIAC-specific master regulatory transcription factors control HNF4A via three distal enhancers to promote GIAC cell proliferation and survival. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/13/2722/F1.large.jpg.


Assuntos
Adenocarcinoma/patologia , Biomarcadores Tumorais/metabolismo , Epigenômica , Neoplasias Gastrointestinais/patologia , Regulação Neoplásica da Expressão Gênica , Fator 4 Nuclear de Hepatócito/metabolismo , Fatores de Transcrição/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Animais , Apoptose , Biomarcadores Tumorais/genética , Proliferação de Células , Neoplasias Gastrointestinais/genética , Neoplasias Gastrointestinais/metabolismo , Redes Reguladoras de Genes , Genômica , Fator 4 Nuclear de Hepatócito/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Prognóstico , Regiões Promotoras Genéticas , Taxa de Sobrevida , Fatores de Transcrição/genética , Transcriptoma , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Environ Sci Process Impacts ; 22(3): 824-832, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-32159184

RESUMO

In exploration of congenital heart defects produced by TCE, Hepatocyte Nuclear Factor 4 alpha (HNF4a) transcriptional activity was identified as a central component. TCE exposure altered gene transcription in the chick heart in a non-monotonic pattern where only low dose exposure inhibited transcription by HNF4a. As the chick embryo is non-placental, we examine here HNF4a as a target of TCE in developing mouse embryos. Benfluorex and Bi6015, published agonist and antagonist, respectively, of HNF4a were compared to low dose TCE exposure. Pregnant mice were exposed to 10 ppb (76 nM) TCE, 5 µM Benfluorex, 5 µM Bi6015, or a combination of Bi6015 and TCE in drinking water. Litters (E12) were collected during a sensitive window in heart development. Embryonic hearts were collected, pooled for extraction of RNA and marker expression was examined by quantitative PCR. Multiple markers, previously identified as sensitive to TCE exposure in chicks or as published targets of HNF4a transcription were significantly affected by Benfluorex, Bi6015 and TCE. Activity of TCE and both HNF4a-specific reagents on transcription argues that HNF4a is a component of TCE cardiotoxicity and likely a proximal target of low dose exposure during development. The effectiveness of these reagents after delivery in maternal drinking water suggests that neither maternal metabolism, nor placental transport is protective of exposure.


Assuntos
Tricloroetileno/toxicidade , Animais , Feminino , Coração/embriologia , Fator 4 Nuclear de Hepatócito/genética , Camundongos , Gravidez
12.
PLoS One ; 15(3): e0230450, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32210451

RESUMO

Hepatocellular carcinoma (HCC) is one of the most common human cancers, its prevalence and severity need us to discover novel early diagnostic biomarkers and new therapeutic strategies. MicroRNA-122 is the most abundant microRNA in the liver, and acts as a tumor suppressor and represses HCC development. In our study we showed that HNF-4α and MiR-122 were down-regulated significantly in hepatocellular carcinoma. Over-expression of HNF-4α inhibit hepatocellular carcinoma cells proliferation. And miR-122 is one of the downstream effector of HNF-4α. Up-regulated miR-122 inhibited hepatocellular carcinoma cells proliferation through regulating ADAM17. Collectively, our results suggested that HNF-4α could inhibit hepatocellular carcinoma proliferation with miR-122 being a downstream target of it. And miR-122 would inhibit hepatocellular carcinoma proliferation by regulating ADAM17 signal pathway.


Assuntos
Proteína ADAM17/genética , Carcinoma Hepatocelular/genética , Fator 4 Nuclear de Hepatócito/genética , Neoplasias Hepáticas/genética , MicroRNAs/genética , Carcinoma Hepatocelular/patologia , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica/genética , Células Hep G2 , Humanos , Fígado/metabolismo , Fígado/patologia , Neoplasias Hepáticas/patologia , Transdução de Sinais/genética , Ativação Transcricional/genética
13.
Int J Mol Sci ; 21(3)2020 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-32023898

RESUMO

Hepatitis B virus (HBV) infection is a major factor in the development of various liver diseases such as hepatocellular carcinoma (HCC). Among HBV encoded proteins, HBV X protein (HBx) is known to play a key role in the development of HCC. Hepatocyte nuclear factor 4α (HNF4α) is a nuclear transcription factor which is critical for hepatocyte differentiation. However, the expression level as well as its regulatory mechanism in HBV infection have yet to be clarified. Here, we observed the suppression of HNF4α in cells which stably express HBV whole genome or HBx protein alone, while transient transfection of HBV replicon or HBx plasmid had no effect on the HNF4α level. Importantly, in the stable HBV- or HBx-expressing hepatocytes, the downregulated level of HNF4α was restored by inhibiting the ERK signaling pathway. Our data show that HNF4α was suppressed during long-term HBV infection in cultured HepG2-NTCP cells as well as in a mouse model following hydrodynamic injection of pAAV-HBV or in mice intravenously infected with rAAV-HBV. Importantly, HNF4α downregulation increased cell proliferation, which contributed to the formation and development of tumor in xenograft nude mice. The data presented here provide proof of the effect of HBV infection in manipulating the HNF4α regulatory pathway in HCC development.


Assuntos
Carcinoma Hepatocelular/virologia , Hepatite B/metabolismo , Fator 4 Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/metabolismo , Neoplasias Hepáticas/virologia , Transativadores/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Modelos Animais de Doenças , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Células Hep G2 , Hepatite B/genética , Hepatite B/virologia , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Camundongos , Camundongos Nus , Transplante de Neoplasias
14.
Nat Commun ; 11(1): 342, 2020 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-31953394

RESUMO

Precise control of hepatic glucose production (HGP) is pivotal to maintain systemic glucose homeostasis. HNF4α functions to stimulate transcription of key gluconeogenic genes. HNF4α harbors two promoters (P2 and P1) thought to be primarily active in fetal and adult livers, respectively. Here we report that the fetal version of HNF4α is required for HGP in the adult liver. This isoform is acutely induced upon fasting and chronically increased in type-2 diabetes (T2D). P2 isoform induction occurs in response to glucagon-stimulated upregulation of TET3, not previously shown to be involved in HGP. TET3 is recruited to the P2 promoter by FOXA2, leading to promoter demethylation and increased transcription. While TET3 overexpression augments HGP, knockdown of either TET3 or the P2 isoform alone in the liver improves glucose homeostasis in dietary and genetic mouse models of T2D. These studies unmask an unanticipated, conserved regulatory mechanism in HGP and offer potential therapeutic targets for T2D.


Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Dioxigenases/metabolismo , Fator 4 Nuclear de Hepatócito/metabolismo , Fígado/metabolismo , Isoformas de Proteínas/metabolismo , Animais , Desmetilação do DNA , Metilação de DNA , Proteínas de Ligação a DNA/metabolismo , Dioxigenases/genética , Modelos Animais de Doenças , Jejum , Regulação da Expressão Gênica , Glucagon/metabolismo , Glucose/metabolismo , Fator 3-beta Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Regiões Promotoras Genéticas , Isoformas de Proteínas/genética , Ativação Transcricional , Transcriptoma , Regulação para Cima
15.
Acta Pharmacol Sin ; 41(2): 278-285, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31554961

RESUMO

Epigallocatechin gallate (EGCG), a major polyphenol in green tea, exhibits diverse biological activities. Previous studies show that EGCG could effectively suppress HBV gene expression and replication, but the role of EGCG in HBV replication and its underlying mechanisms, especially the signaling pathways involved, remain unclear. In this study we investigated the mechanisms underlying EGCG inhibition on HBV replication with a focus on the signaling pathways. We showed that EGCG (12.5-50 µM) dose-dependently inhibited HBV gene expression and replication in HepG2.2.15 cells. Similar results were observed in HBV mice receiving EGCG (25 mg· kg-1· d-1, ip) for 5 days. In HepG2.2.15 cells, we showed that EGCG (12.5-50 µM) significantly activate ERK1/2 MAPK signaling, slightly activate p38 MAPK and JAK2/STAT3 signaling, while had no significant effect on the activation of JNK MAPK, PI3K/AKT/mTOR and NF-κB signaling. By using specific inhibitors of these signaling pathways, we demonstrated that ERK1/2 signaling pathway, but not other signaling pathways, was involved in EGCG-mediated inhibition of HBV transcription and replication. Furthermore, we showed that EGCG treatment dose-dependently decreased the expression of hepatocyte nuclear factor 4α (HNF4α) both at the mRNA and protein levels, which could be reversed by pretreatment with the ERK1/2 inhibitor PD98059 (20 µM). Moreover, we revealed that EGCG treatment dose-dependently inhibited the activity of HBV core promoter and the following HBV replication. In summary, our results demonstrate that EGCG inhibits HBV gene expression and replication, which involves ERK1/2-mediated downregulation of HNF4α.These data reveal a novel mechanism for EGCG to inhibit HBV gene expression and replication.


Assuntos
Catequina/análogos & derivados , Vírus da Hepatite B/efeitos dos fármacos , Hepatite B/tratamento farmacológico , Replicação Viral/efeitos dos fármacos , Animais , Antivirais/administração & dosagem , Antivirais/farmacologia , Catequina/administração & dosagem , Catequina/farmacologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Células Hep G2 , Hepatite B/genética , Hepatite B/virologia , Fator 4 Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo
16.
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
17.
Oncogene ; 39(7): 1572-1589, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31695151

RESUMO

Hepatocyte nuclear factor 4α (HNF4α, NR2A1) is a highly conserved member of the nuclear receptor superfamily. Recent advances reveal that it is a key transcriptional regulator of genes, broadly involved in xenobiotic and drug metabolism and also cancers of gastrointestinal tract. However, the exact functional roles of HNF4α in prostate cancer progression are still not fully understood. In this study, we determined the functional significance of HNF4α in prostate cancer. Our results showed that HNF4α exhibited a reduced expression pattern in clinical prostate cancer tissues, prostate cancer cell lines and xenograft model of castration-relapse prostate cancer. Stable HNF4α knockdown not only could promote cell proliferation and suppress doxorubicin (Dox)-induced cellular senescence in prostate cancer cells, but also confer resistance to paclitaxel treatment and enhance colony formation capacity and in vivo tumorigenicity of prostate cancer cells. On the contrary, ectopic overexpression of HNF4α could significantly inhibit the cell proliferation of prostate cancer cells, induce cell-cycle arrest at G2/M phase and trigger the cellular senescence in prostate cancer cells by activation of p21 signal pathway in a p53-independent manner via its direct transactivation of CDKN1A. Together, our results show that HNF4α performs a tumor suppressor function in prostate cancer via a mechanism of p21-driven cellular senescence.


Assuntos
Senescência Celular , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Fator 4 Nuclear de Hepatócito/metabolismo , Neoplasias da Próstata/metabolismo , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Epigênese Genética , Células Epiteliais/patologia , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Genes ras/genética , Fator 4 Nuclear de Hepatócito/deficiência , Fator 4 Nuclear de Hepatócito/genética , Humanos , Masculino , Neoplasias da Próstata/patologia
18.
Dig Dis Sci ; 65(7): 2009-2023, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-31722057

RESUMO

BACKGROUND AND AIMS: Bioartificial livers (BALs) have attracted much attention as potential supportive therapies for liver diseases. A serum-free microcarrier culture strategy for the in vitro high-density expansion of human-induced hepatocyte-like cells (hiHeps) suitable for BALs was studied in this article. METHODS: hiHeps were transdifferentiated from human fibroblasts by the lentiviral overexpression of FOXA3, HNF1A, and HNF4A. Cells were cultured on microcarriers, their proliferation was evaluated by cell count and CCK-8 assays, and their function was evaluated by detecting liver function parameters in the supernatant, including urea secretion, albumin synthesis, and lactate dehydrogenase levels. The expressions of hepatocyte function-associated genes of hiHeps were measured by qRT-PCR in 2D and 3D conditions. The expression of related proteins during fibronectin promotes cell adhesion, and proliferation on microcarrier was detected by western blotting. RESULTS: During microcarrier culture, the optimal culture conditions during the adherence period were the use of half-volume high-density inoculation, Cytodex 3 at a concentration of 3 mg/mL, a cell seeding density of 2.0 × 105 cells/mL, and a stirring speed of 45 rpm. The final cell density in self-developed, chemically defined serum-free medium (SFM) reached 2.53 × 106 cells/mL, and the maximum increase in expansion was 12.61-fold. In addition, we found that fibronectin (FN) can promote hiHep attachment and proliferation on Cytodex 3 microcarriers and that this pro-proliferative effect was mediated by the integrin-ß1/FAK/ERK/CyclinD1 signaling pathway. Finally, the growth and function of hiHeps on Cytodex 3 in SFM were close to those of hiHeps on Cytodex 3 in hepatocyte maintenance medium (HMM), and cells maintained their morphology and function after harvest on microcarriers. CONCLUSIONS: Serum-free microcarrier culture has important implications for the expansion of a sufficient number of hiHeps prior to the clinical application of BALs.


Assuntos
Técnicas de Cultura de Células/métodos , Proliferação de Células , Transdiferenciação Celular , Hepatócitos/citologia , Fígado Artificial , Albuminas/biossíntese , Adesão Celular , Técnicas de Reprogramação Celular/métodos , Meios de Cultura Livres de Soro , Ciclina D1/metabolismo , Dextranos , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibronectinas/metabolismo , Quinase 1 de Adesão Focal/metabolismo , Fator 1-alfa Nuclear de Hepatócito/genética , Fator 3-gama Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/genética , Hepatócitos/metabolismo , Hepatócitos/fisiologia , Humanos , Integrina beta1/metabolismo , L-Lactato Desidrogenase/metabolismo , Sistema de Sinalização das MAP Quinases , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ureia/metabolismo
19.
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
20.
PLoS Genet ; 15(12): e1008508, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31815936

RESUMO

Zinc is essential for cellular functions as it is a catalytic and structural component of many proteins. In contrast, cadmium is not required in biological systems and is toxic. Zinc and cadmium levels are closely monitored and regulated as their excess causes cell stress. To maintain homeostasis, organisms induce metal detoxification gene programs through stress responsive transcriptional regulatory complexes. In Caenorhabditis elegans, the MDT-15 subunit of the evolutionarily conserved Mediator transcriptional coregulator is required to induce genes upon exposure to excess zinc and cadmium. However, the regulatory partners of MDT-15 in this response, its role in cellular and physiological stress adaptation, and the putative role for mammalian MED15 in the metal stress responses remain unknown. Here, we show that MDT-15 interacts physically and functionally with the Nuclear Hormone Receptor HIZR-1 to promote molecular, cellular, and organismal adaptation to cadmium and excess zinc. Using gain- and loss-of-function mutants and qRT-PCR and reporter analysis, we find that mdt-15 and hizr-1 cooperate to induce zinc and cadmium responsive genes. Moreover, the two proteins interact physically in yeast-two-hybrid assays and this interaction is enhanced by the addition of zinc or cadmium, the former a known ligand of HIZR-1. Functionally, mdt-15 and hizr-1 mutants show defective storage of excess zinc in the gut and are hypersensitive to zinc-induced reductions in egg-laying. Furthermore, mdt-15 but not hizr-1 mutants are hypersensitive to cadmium-induced reductions in egg-laying, suggesting potential divergence of regulatory pathways. Lastly, mammalian MDT-15 orthologs bind genomic regulatory regions of metallothionein and zinc transporter genes in a cadmium and zinc-stimulated fashion, and human MED15 is required to induce a metallothionein gene in lung adenocarcinoma cells exposed to cadmium. Collectively, our data show that mdt-15 and hizr-1 cooperate to regulate cadmium detoxification and zinc storage and that this mechanism is at least partially conserved in mammals.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Fator 4 Nuclear de Hepatócito/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Zinco/toxicidade , Animais , Caenorhabditis elegans/efeitos dos fármacos , Proteínas de Caenorhabditis elegans/genética , Proteínas de Transporte/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Fator 4 Nuclear de Hepatócito/genética , Humanos , Metalotioneína/genética , Mutação , Regiões Promotoras Genéticas , Receptores Citoplasmáticos e Nucleares/genética , Estresse Fisiológico , Fatores de Transcrição/genética , Técnicas do Sistema de Duplo-Híbrido
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