RESUMO
BACKGROUND & AIMS: Functions of intestinal stem cells (ISCs) are regulated by diet and metabolic pathways. Hepatocyte nuclear factor 4 (HNF4) family are transcription factors that bind fatty acids. We investigated how HNF4 transcription factors regulate metabolism and their functions in ISCs in mice. METHODS: We performed studies with Villin-CreERT2;Lgr5-EGFP-IRES-CreERT2;Hnf4αf/f;Hnf4γCrispr/Crispr mice, hereafter referred to Hnf4αγDKO. Mice were given tamoxifen to induce Cre recombinase. Mice transgenic with only Cre alleles (Villin-CreERT2, Lgr5-EGFP-IRES-CreERT2, Hnf4α+/+, and Hnf4γ+/+) or mice given vehicle were used as controls. Crypt and villus cells were isolated, incubated with fluorescently labeled fatty acids or glucose analog, and analyzed by confocal microscopy. Fatty acid oxidation activity and tricarboxylic acid (TCA) cycle metabolites were measured in cells collected from the proximal half of the small intestine of Hnf4αγDKO and control mice. We performed chromatin immunoprecipitation and gene expression profiling analyses to identify genes regulated by HNF4 factors. We established organoids from duodenal crypts, incubated them with labeled palmitate or acetate, and measured production of TCA cycle metabolites or fatty acids. Acetate, a precursor of acetyl coenzyme A (CoA) (a product of fatty acid ß-oxidation [FAO]), or dichloroacetate, a compound that promotes pyruvate oxidation and generation of mitochondrial acetyl-CoA, were used for metabolic intervention. RESULTS: Crypt cells rapidly absorbed labeled fatty acids, and messenger RNA levels of Lgr5+ stem cell markers (Lgr5, Olfm4, Smoc2, Msi1, and Ascl2) were down-regulated in organoids incubated with etomoxir, an inhibitor of FAO, indicating that FAO was required for renewal of ISCs. HNF4A and HNF4G were expressed in ISCs and throughout the intestinal epithelium. Single knockout of either HNF4A or HNF4G did not affect maintenance of ISCs, but double-knockout of HNF4A and HNF4G resulted in ISC loss; stem cells failed to renew. FAO supports ISC renewal, and HNF4 transcription factors directly activate FAO genes, including Acsl5 and Acsf2 (encode regulators of acyl-CoA synthesis), Slc27a2 (encodes a fatty acid transporter), Fabp2 (encodes fatty acid binding protein), and Hadh (encodes hydroxyacyl-CoA dehydrogenase). In the intestinal epithelium of Hnf4αγDKO mice, expression levels of FAO genes, FAO activity, and metabolites of TCA cycle were all significantly decreased, but fatty acid synthesis transcripts were increased, compared with control mice. The contribution of labeled palmitate or acetate to the TCA cycle was reduced in organoids derived from Hnf4αγDKO mice, compared with control mice. Incubation of organoids derived from double-knockout mice with acetate or dichloroacetate restored stem cells. CONCLUSIONS: In mice, the transcription factors HNF4A and HNF4G regulate the expression of genes required for FAO and are required for renewal of ISCs.
Assuntos
Ácidos Graxos/metabolismo , Fator 4 Nuclear de Hepatócito/fisiologia , Intestino Delgado/citologia , Células-Tronco/metabolismo , Animais , Duodeno/citologia , Proteínas de Ligação a Ácido Graxo/metabolismo , Mucosa Intestinal/citologia , Camundongos , Camundongos Knockout , Organoides/metabolismo , OxirreduçãoRESUMO
BACKGROUND AND AIMS: Telomere attrition is a major risk factor for end-stage liver disease. Due to a lack of adequate models and intrinsic difficulties in studying telomerase in physiologically relevant cells, the molecular mechanisms responsible for liver disease in patients with telomere syndromes remain elusive. To circumvent that, we used genome editing to generate isogenic human embryonic stem cells (hESCs) harboring clinically relevant mutations in telomerase and subjected them to an in vitro, stage-specific hepatocyte differentiation protocol that resembles hepatocyte development in vivo. APPROACH AND RESULTS: Using this platform, we observed that while telomerase is highly expressed in hESCs, it is quickly silenced, specifically due to telomerase reverse transcriptase component (TERT) down-regulation, immediately after endoderm differentiation and completely absent in in vitro-derived hepatocytes, similar to what is observed in human primary hepatocytes. While endoderm derivation is not impacted by telomere shortening, progressive telomere dysfunction impaired hepatic endoderm formation. Consequently, hepatocyte derivation, as measured by expression of specific hepatic markers as well by albumin expression and secretion, is severely compromised in telomerase mutant cells with short telomeres. Interestingly, this phenotype was not caused by cell death induction or senescence. Rather, telomere shortening prevents the up-regulation and activation of human hepatocyte nuclear factor 4 alpha (HNF4α) in a p53-dependent manner. Both reactivation of telomerase and silencing of p53 rescued hepatocyte formation in telomerase mutants. Likewise, the conditional expression (doxycycline-controlled) of HNF4α, even in cells that retained short telomeres, accrued DNA damage, and exhibited p53 stabilization, successfully restored hepatocyte formation from hESCS. CONCLUSIONS: Our data show that telomere dysfunction acts as a major regulator of HNF4α during hepatocyte development, pointing to a target in the treatment of liver disease in telomere-syndrome patients.
Assuntos
Fator 4 Nuclear de Hepatócito/fisiologia , Hepatócitos/fisiologia , Telômero/fisiologia , Proteína Supressora de Tumor p53/fisiologia , Diferenciação Celular , Células Cultivadas , Células-Tronco Embrionárias , Hepatócitos/citologia , Humanos , Telomerase/genéticaRESUMO
Organ liver transplantation and hepatocyte transplantation are not performed to their full potential because of donor shortage, which could be resolved by identifying new donor sources for the development of hepatocyte-like cells (HLCs). HLCs have been differentiated from some stem cell sources as alternative primary hepatocytes throughout the world; however, the currently available techniques cannot differentiate HLCs to the level of normal adult primary hepatocytes. The outstanding questions are as follows: which stem cells are the best cell sources? which protocol is the best way to differentiate them into HLCs? what is the definition of differentiated HLCs? how can we enforce the function of HLCs? what is the difference between HLCs and primary hepatocytes? what are the problems with HLC transplantation? This review summarizes the current status of HLCs, focusing on stem cell sources, the differentiation protocol for HLCs, the general characterization of HLCs, the generation of more functional HLCs, comparison with primary hepatocytes, and HLCs in cell-transplantation-based liver regeneration.
Assuntos
Diferenciação Celular , Terapia Baseada em Transplante de Células e Tecidos/métodos , Técnicas Citológicas/métodos , Hepatócitos/transplante , Hepatopatias/terapia , Células-Tronco/fisiologia , Proteínas Morfogenéticas Ósseas/fisiologia , Diferenciação Celular/genética , Células Cultivadas , Técnicas de Cocultura , Fatores de Crescimento de Fibroblastos/fisiologia , Fator 4 Nuclear de Hepatócito/fisiologia , Proteínas de Homeodomínio/fisiologia , Humanos , Regeneração Hepática/fisiologia , Fatores de Transcrição SOXF/fisiologia , Fatores de Transcrição/fisiologiaRESUMO
Hepatocyte nuclear factor 4 (HNF4) plays essential roles in regulating lipid metabolism and glucose homeostasis in female insects. However, little is known about the role of HNF4 in insect fecundity. Here we demonstrate that HNF4 regulates female fecundity by affecting egg hatching in the brown planthopper (BPH) Nilaparvata lugens. HNF4 was highly expressed in the ovary and fat body of female adult. RNA interference-mediated HNF4 knockdown resulted in a dramatic reduction in egg hatchability and caused a severe block in embryonic development, while showed no significant effects on ovary development and egg laying. Transcriptome sequencing analysis showed that 72 genes encoding ribosome proteins were significantly down-regulated in the HNF4-silenced BPH and "ribosome" was the most-enriched pathway for the down-regulated genes. These results suggest that HNF4 controls the dynamics of egg structure, likely through its regulation of ribosome protein genes, which in turn affects the embryonic development and egg hatching.
Assuntos
Hemípteros/genética , Fator 4 Nuclear de Hepatócito/fisiologia , Proteínas de Insetos/fisiologia , Animais , Feminino , Fertilidade/genética , Hemípteros/embriologia , Hemípteros/crescimento & desenvolvimento , Hemípteros/metabolismo , Fator 4 Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/metabolismo , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Masculino , Ovário/metabolismo , Interferência de RNA , RNA-Seq , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , TranscriptomaRESUMO
Objective: To explore the role and molecular mechanism of hepatocyte nuclear factor 4γ (HNF4γ) in proliferation and stemness of gastric cancer. Methods: A total of 102 cases of paraffin-embedded gastric cancer tissues and matched adjacent gastric tissues and 42 cases of fresh-frozen tissues derived from gastric patients who received radical gastrectomy were collected from the First Affiliated Hospital of Zhengzhou University between 2012 to 2015. The expression of HNF4γ was tested by immunohistochemical staining, quantitative real-time polymerase chain reaction (qRT-PCR). HNF4γ overexpressed (AGS-HNF4γ) and shRNA silenced (HGC27-shHNF4γ) gastric cell lines were established. The effects of HNF4γ on cell proliferation and stemness were verified by XTT, clone formation and sphere formation assay. The expression of CD44 was detected by western blot. Results: The mRNA expression level of HNF4γ in fresh-frozen gastric cancer tissue was (12.43±2.702), which was significantly higher than (3.639±1.109) in normal tissue (P<0.001). The high protein expression rate of HNF4γ in paraffin-embedded gastric cancer tissues was 41.2% (42/102), which was significantly higher than 8.8% (9/102) in normal gastric mucosa tissue (P< 0.001). The protein expression of HNF4γ was closely related to the tumor differentiation, infiltration depth, lymph node metastasis and tumor stage (P<0.05). The median survival interval of patients with HNF4γ high expression was 25 months, the 3-year survival rate was 4.8% (2/42), significantly lower than 38 months and 51.7% (31/60) of patients with normal HNF4γ expression (P<0.001). The proliferation and CD44 protein expression of AGS-HNF4γ cells were significantly higher than those of the AGS-Vector cells. The number of clone formation, sphere formation rate of AGS-HNF4γ cells were 243.5±24.5 and (83.5±3.9)%, significantly higher than 81.0±16.0 and (21.8±5.6)% of AGS-Vector cells (P=0.030 and P=0.010, respectively). The proliferation and CD44 protein expression of HGC27-shHNF4 cells were significantly lower than those of the HGC27-vector cells. The number of clone formation, sphere formation rate of HGC27-shHNF4 cells were 26.0±1.0 and (20.8±8.4)%, significantly higher than 83.5±4.5 and (72.5±4.8)% of HGC27-vector cells (P=0.006 and P=0.030, respectively). Conclusions: HNF4γ is upregulated in the gastric cancer tissues and related with the poor prognosis of patients with gastric cancer. Overexpression of HNF4γ promotes the proliferation and remains the stemness of gastric cancer cells by upregulating the expression of CD44.
Assuntos
Carcinoma , Fator 4 Nuclear de Hepatócito/fisiologia , Neoplasias Gástricas , Linhagem Celular Tumoral , Proliferação de Células , Gastrectomia , Regulação Neoplásica da Expressão Gênica , Fator 4 Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/metabolismo , Fatores Nucleares de Hepatócito , Humanos , Prognóstico , Neoplasias Gástricas/genética , Neoplasias Gástricas/cirurgiaRESUMO
HNF4α (hepatocyte nuclear factor 4α) is one of the master regulators of pancreatic ß-cell development and function, and mutations in the HNF4α gene are well-known monogenic causes of diabetes. As a member of the nuclear receptor family, HNF4α exerts its gene regulatory function through various molecular interactions; however, there is a paucity of knowledge of the different functional complexes in which HNF4α participates. Here, to find HNF4α-binding proteins in pancreatic ß-cells, we used yeast two-hybrid screening, a mammalian two-hybrid assay, and glutathione S-transferase pulldown approaches, which identified EBP1 (ErbB3-binding protein 1) as a factor that binds HNF4α in a LXXLL motif-mediated manner. In the ß-cells, EBP1 suppressed the expression of HNF4α target genes that are implicated in insulin secretion, which is impaired in HNF4α mutation-driven diabetes. The crystal structure of the HNF4α ligand-binding domain in complex with a peptide harboring the EBP1 LXXLL motif at 3.15Å resolution hinted at the molecular basis of the repression. The details of the structure suggested that EBP1's LXXLL motif competes with HNF4α coactivators for the same binding pocket and thereby prevents recruitment of additional transcriptional coactivators. These findings provide further evidence that EBP1 plays multiple cellular roles and is involved in nuclear receptor-mediated gene regulation. Selective disruption of the HNF4α-EBP1 interaction or tissue-specific EBP1 inactivation can enhance HNF4α activities and thereby improve insulin secretion in ß-cells, potentially representing a new strategy for managing diabetes and related metabolic disorders.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fator 4 Nuclear de Hepatócito/metabolismo , Células Secretoras de Insulina/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Proteínas de Transporte/metabolismo , Proteínas de Ligação a DNA/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Regulação da Expressão Gênica/genética , Células HeLa , Fator 4 Nuclear de Hepatócito/fisiologia , Humanos , Insulina/metabolismo , Secreção de Insulina/genética , Células Secretoras de Insulina/fisiologia , Regiões Promotoras Genéticas/genética , Ligação Proteica/fisiologia , Proteínas de Ligação a RNA/fisiologia , Fatores de TranscriçãoRESUMO
Hepatocyte nuclear factor 4 alpha (HNF4α) is critical for hepatic differentiation. Recent studies have highlighted its role in inhibition of hepatocyte proliferation and tumor suppression. However, the role of HNF4α in liver regeneration (LR) is not known. We hypothesized that hepatocytes modulate HNF4α activity when navigating between differentiated and proliferative states during LR. Western blotting analysis revealed a rapid decline in nuclear and cytoplasmic HNF4α protein levels, accompanied with decreased target gene expression, within 1 hour after two-thirds partial hepatectomy (post-PH) in C57BL/6J mice. HNF4α protein expression did not recover to pre-PH levels until day 3. Hepatocyte-specific deletion of HNF4α (HNF4α-KO [knockout]) in mice resulted in 100% mortality post-PH, despite increased proliferative marker expression throughout regeneration. Sustained loss of HNF4α target gene expression throughout regeneration indicated that HNF4α-KO mice were unable to compensate for loss of HNF4α transcriptional activity. Deletion of HNF4α resulted in sustained proliferation accompanied by c-Myc and cyclin D1 overexpression and a complete deficiency of hepatocyte function after PH. Interestingly, overexpression of degradation-resistant HNF4α in hepatocytes delayed, but did not prevent, initiation of regeneration after PH. Finally, adeno-associated virus serotype 8 (AAV8)-mediated reexpression of HNF4α in hepatocytes of HNF4α-KO mice post-PH restored HNF4α protein levels, induced target gene expression, and improved survival of HNF4α-KO mice post-PH. Conclusion: In conclusion, these data indicate that HNF4α reexpression following initial decrease is critical for hepatocytes to exit from cell cycle and resume function during the termination phase of LR. These results indicate the role of HNF4α in LR and have implications for therapy of liver failure.
Assuntos
Fator 4 Nuclear de Hepatócito/fisiologia , Regeneração Hepática/fisiologia , Animais , Hepatócitos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
LncRNAs play critical roles in various pathophysiological and biological processes, such as protein translation, RNA splicing, and epigenetic modification. Indeed, abundant evidences demonstrated that lncRNA act as competing endogenous RNAs (ceRNAs) to participate in tumorigenesis. However, little is known about the underlying function of lncRNA in nonhomologous end joining (NHEJ) pathway 1 (LINP1) in pediatric and adolescent acute myeloid leukemia (AML). The expression of LINP1 was examined in AML patient samples by qRT-PCR. Cell proliferation was examined by CCK-8 and Edu assays. ß-Galactosidase senescence assay, mGlucose uptake assay, lactate production assay, and Gene Ontology (GO) analysis were performed for functional analysis. We found that LINP1 was significantly overexpressed in AML patients at diagnosis, whereas downregulated after complete remission (CR). Furthermore, knockdown of LINP1 expression remarkably suppressed glucose uptake and AML cell maintenance. Mechanistically, LINP1 was found to inhibit the glucose metabolism by suppressing the expression of HNF4a. Both LINP1 and HNF4a knockdown reduced the expression levels of AMPK phosphorylation and WNT5A, indicating for the first time that LINP1 strengthened the HNF4a-AMPK/WNT5A signaling pathway involved in cell glucose metabolism modulation and AML cell survival. Taken together, our results indicated that LINP1 promotes the malignant phenotype of AML cells and stimulates glucose metabolism, which can be regarded as a potential prognostic marker and therapeutic target for AML.
Assuntos
Adenilato Quinase/fisiologia , Fator 4 Nuclear de Hepatócito/fisiologia , Leucemia Mieloide Aguda/genética , RNA Longo não Codificante/fisiologia , RNA Neoplásico/fisiologia , Transdução de Sinais/fisiologia , Proteína Wnt-5a/fisiologia , Adolescente , Animais , Medula Óssea/patologia , Divisão Celular , Criança , Regulação Leucêmica da Expressão Gênica , Técnicas de Silenciamento de Genes , Ontologia Genética , Glucose/metabolismo , Fator 4 Nuclear de Hepatócito/biossíntese , Fator 4 Nuclear de Hepatócito/genética , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Transplante de Neoplasias , Púrpura Trombocitopênica Idiopática/metabolismo , Interferência de RNA , RNA Longo não Codificante/biossíntese , RNA Longo não Codificante/genética , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , RNA Interferente Pequeno/genética , Distribuição Aleatória , Indução de Remissão , Transdução de Sinais/genética , Células THP-1RESUMO
Androgen receptor (AR) binds male sex steroids and mediates physiological androgen actions in target tissues. ChIP-seq analyses of AR-binding events in murine prostate, kidney and epididymis show that in vivo AR cistromes and their respective androgen-dependent transcription programs are highly tissue specific mediating distinct biological pathways. This high order of tissue specificity is achieved by the use of exclusive collaborating factors in the three androgen-responsive tissues. We find two novel collaborating factors for AR signaling in vivo--Hnf4α (hepatocyte nuclear factor 4α) in mouse kidney and AP-2α (activating enhancer binding protein 2α) in mouse epididymis--that define tissue-specific AR recruitment. In mouse prostate, FoxA1 serves for the same purpose. FoxA1, Hnf4α and AP-2α motifs are over-represented within unique AR-binding loci, and the cistromes of these factors show substantial overlap with AR-binding events distinct to each tissue type. These licensing or pioneering factors are constitutively bound to chromatin and guide AR to specific genomic loci upon hormone exposure. Collectively, liganded receptor and its DNA-response elements are required but not sufficient for establishment of tissue-specific transcription programs.
Assuntos
Epididimo/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Fator 3-alfa Nuclear de Hepatócito/fisiologia , Fator 4 Nuclear de Hepatócito/fisiologia , Rim/metabolismo , Próstata/metabolismo , Receptores Androgênicos/metabolismo , Testosterona/farmacologia , Fator de Transcrição AP-2/fisiologia , Animais , Linhagem Celular , Cromatina/metabolismo , Imunoprecipitação da Cromatina , Epididimo/citologia , Perfilação da Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos ICR , Orquiectomia , Especificidade de Órgãos , Ligação Proteica , Interferência de RNA , RNA Interferente Pequeno/farmacologia , Proteínas de Ligação a Tacrolimo/biossíntese , Proteínas de Ligação a Tacrolimo/genética , Testosterona/fisiologia , Transcrição GênicaRESUMO
Immortalized human hepatocytes (IHH) could provide an unlimited supply of hepatocytes, but insufficient differentiation and phenotypic instability restrict their clinical application. This study aimed to determine the role of hepatocyte nuclear factor 4A (HNF4A) in hepatic differentiation of IHH, and whether encapsulation of IHH overexpressing HNF4A could improve liver function and survival in rats with acute liver failure (ALF). Primary human hepatocytes were transduced with lentivirus-mediated catalytic subunit of human telomerase reverse transcriptase (hTERT) to establish IHH. Cells were analyzed for telomerase activity, proliferative capacity, hepatocyte markers, and tumorigenicity (c-myc) expression. Hepatocyte markers, hepatocellular functions, and morphology were studied in the HNF4A-overexpressing IHH. Hepatocyte markers and karyotype analysis were completed in the primary hepatocytes using shRNA knockdown of HNF4A. Nuclear translocation of ß-catenin was assessed. Rat models of ALF were treated with encapsulated IHH or HNF4A-overexpressing IHH. A HNF4A-positive IHH line was established, which was non-tumorigenic and conserved properties of primary hepatocytes. HNF4A overexpression significantly enhanced mRNA levels of genes related to hepatic differentiation in IHH. Urea levels were increased by the overexpression of HNF4A, as measured 24h after ammonium chloride addition, similar to that of primary hepatocytes. Chromosomal abnormalities were observed in primary hepatocytes transfected with HNF4A shRNA. HNF4α overexpression could significantly promote ß-catenin activation. Transplantation of HNF4A overexpressing IHH resulted in better liver function and survival of rats with ALF compared with IHH. HNF4A improved hepatic differentiation of IHH. Transplantation of HNF4A-overexpressing IHH could improve the liver function and survival in a rat model of ALF.
Assuntos
Diferenciação Celular/genética , Fator 4 Nuclear de Hepatócito/fisiologia , Hepatócitos/fisiologia , Hepatócitos/transplante , Falência Hepática Aguda/terapia , Fígado/fisiologia , Adulto , Animais , Linhagem Celular Transformada , Sobrevivência Celular/genética , Células Cultivadas , Hepatócitos/patologia , Humanos , Falência Hepática Aguda/genética , Falência Hepática Aguda/patologia , Transplante de Fígado/métodos , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
The transcription factor, X-box-binding protein-1 (XBP1), controls the development and maintenance of the endoplasmic reticulum (ER) in multiple secretory cell lineages. We show here that Hepatocyte Nuclear Factor 4α (HNF4α) directly induces XBP1 expression. Mutations in HNF4α cause Mature-Onset Diabetes of the Young I (MODYI), a subset of diabetes characterized by diminished GSIS. In mouse models, cell lines, and ex vivo islets, using dominant negative and human- disease-allele point mutants or knock-out and knockdown models, we show that disruption of HNF4α caused decreased expression of XBP1 and reduced cellular ER networks. GSIS depends on ER Ca(2+) signaling; we show that diminished XBP1 and/or HNF4α in ß-cells led to impaired ER Ca(2+) homeostasis. Restoring XBP1 expression was sufficient to completely rescue GSIS in HNF4α-deficient ß-cells. Our findings uncover a transcriptional relationship between HNF4α and Xbp1 with potentially broader implications about MODYI and the importance of transcription factor signaling in the regulation of secretion.
Assuntos
Proteínas de Ligação a DNA/genética , Fator 4 Nuclear de Hepatócito/fisiologia , Células Secretoras de Insulina/fisiologia , Fatores de Transcrição/genética , Transcrição Gênica , Animais , Cálcio/metabolismo , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Diabetes Mellitus Tipo 2/genética , Retículo Endoplasmático/metabolismo , Regulação da Expressão Gênica , Glucose/fisiologia , Células HEK293 , Homeostase , Humanos , Insulina/metabolismo , Secreção de Insulina , Camundongos , Fatores de Transcrição de Fator Regulador X , Fatores de Transcrição/metabolismo , Proteína 1 de Ligação a X-BoxRESUMO
BACKGROUND AND AIMS: Epithelial-to-mesenchymal transition (EMT) and the reverse mesenchymal-to-epithelial transition (MET) are manifestations of cellular plasticity that imply a dynamic and profound gene expression reprogramming. While a major epigenetic code controlling the coordinated regulation of a whole transcriptional profile is guaranteed by DNA methylation, DNA methyltransferase (DNMT) activities in EMT/MET dynamics are still largely unexplored. Here, we investigated the molecular mechanisms directly linking HNF4α, the master effector of MET, to the regulation of both de novo of DNMT 3A and 3B. METHODS: Correlation among EMT/MET markers, microRNA29 and DNMT3s expression was evaluated by RT-qPCR, Western blotting and immunocytochemical analysis. Functional roles of microRNAs and DNMT3s were tested by anti-miRs, microRNA precursors and chemical inhibitors. ChIP was utilized for investigating HNF4α DNA binding activity. RESULTS: HNF4α silencing was sufficient to induce positive modulation of DNMT3B, in in vitro differentiated hepatocytes as well as in vivo hepatocyte-specific Hnf4α knockout mice, and DNMT3A, in vitro, but not DNMT1. In exploring the molecular mechanisms underlying these observations, evidence have been gathered for (i) the inverse correlation between DNMT3 levels and the expression of their regulators miR-29a and miR-29b and (ii) the role of HNF4α as a direct regulator of miR-29a-b transcription. Notably, during TGFß-induced EMT, DNMT3s' pivotal function has been proved, thus suggesting the need for the repression of these DNMTs in the maintenance of a differentiated phenotype. CONCLUSIONS: HNF4α maintains hepatocyte identity by regulating miR-29a and -29b expression, which in turn control epigenetic modifications by limiting DNMT3A and DNMT3B levels.
Assuntos
Diferenciação Celular/genética , Transformação Celular Neoplásica/genética , DNA (Citosina-5-)-Metiltransferases/genética , Epigênese Genética/fisiologia , Transição Epitelial-Mesenquimal/genética , Fator 4 Nuclear de Hepatócito/fisiologia , Hepatócitos/citologia , MicroRNAs/fisiologia , Animais , Células Cultivadas , Reprogramação Celular/genética , DNA Metiltransferase 3A , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Hepatócitos/metabolismo , Camundongos , Camundongos KnockoutRESUMO
Hepatitis C virus (HCV) is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma in humans. The life cycle of HCV is closely associated with the metabolism of lipids, especially very-low-density lipoprotein (VLDL) in hepatocytes. Hepatocyte nuclear factor 4α (HNF4α), the most abundant transcription factor in the liver, regulates the VLDL secretory pathway. However, the effects of HNF4α on the HCV life cycle are unclear. In this study, we investigated the regulatory effects of HNF4α on HCV assembly and secretion. HCV in HNF4α-deficient hepatocytes showed reduced assembly and secretion but unchanged entry and RNA replication. Bezafibrate, a chemical inhibitor of HNF4α, suppressed HCV assembly and secretion. HNF4α downregulation resulted in rearrangement of cytosolic lipid droplets (LDs), as evidenced by the aggregation of large LDs and distorted cytosolic distribution. Phospholipase A2 GXIIB (PLA2GXIIB), an HNF4α-regulated factor involved in VLDL secretion, was found to be crucial in HCV secretion. PLA2GXIIB expression was upregulated in hepatocytes harboring HCV subgenomic replicons or in HCV-infected hepatocytes. This upregulation was transcriptionally controlled in an HNF4α-dependent manner after HCV infection. Furthermore, PLA2GXIIB combined with microsomal triglyceride transfer protein was found to be responsible for the regulation of HNF4α-induced HCV infectivity. These results suggest that HNF4α and its downstream PLA2GXIIB are important factors affecting the late stage of the HCV life cycle and may serve as potential drug targets for the treatment of HCV infection.
Assuntos
Hepacivirus/enzimologia , Fator 4 Nuclear de Hepatócito/fisiologia , Fosfolipases A2/metabolismo , Linhagem Celular , Inativação Gênica , Hepacivirus/genética , Hepacivirus/patogenicidade , Hepacivirus/fisiologia , Humanos , Regiões Promotoras Genéticas , RNA Viral/biossíntese , Montagem de Vírus , Replicação ViralRESUMO
Acat2 [gene name: sterol O-acyltransferase 2 (SOAT2)] esterifies cholesterol in enterocytes and hepatocytes. This study aims to identify repressor elements in the human SOAT2 promoter and evaluate their in vivo relevance. We identified TG-interacting factor 1 (Tgif1) to function as an important repressor of SOAT2. Tgif1 could also block the induction of the SOAT2 promoter activity by hepatocyte nuclear factor 1α and 4α. Women have â¼ 30% higher hepatic TGIF1 mRNA compared with men. Depletion of Tgif1 in mice increased the hepatic Soat2 expression and resulted in higher hepatic lipid accumulation and plasma cholesterol levels. Tgif1 is a new player in human cholesterol metabolism.
Assuntos
Inativação Gênica , Proteínas de Homeodomínio/fisiologia , Proteínas Repressoras/fisiologia , Esterol O-Aciltransferase/genética , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Repressão Enzimática , Feminino , Cálculos Biliares/enzimologia , Fator 1-alfa Nuclear de Hepatócito/fisiologia , Fator 4 Nuclear de Hepatócito/fisiologia , Proteínas de Homeodomínio/metabolismo , Humanos , Lipídeos/sangue , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Regiões Promotoras Genéticas , Ligação Proteica , Caracteres Sexuais , Esterol O-Aciltransferase/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Esterol O-Aciltransferase 2RESUMO
Cytochrome P450 2D6 (CYP2D6), a major drug-metabolizing enzyme, is responsible for metabolism of approximately 25% of marketed drugs. Clinical evidence indicates that metabolism of CYP2D6 substrates is increased during pregnancy, but the underlying mechanisms remain unclear. To identify transcription factors potentially responsible for CYP2D6 induction during pregnancy, a panel of genes differentially expressed in the livers of pregnant versus nonpregnant CYP2D6-humanized (tg-CYP2D6) mice was compiled via microarray experiments followed by real-time quantitative reverse-transcription polymerase chain reaction(qRT-PCR) verification. As a result, seven transcription factors-activating transcription factor 5 (ATF5), early growth response 1 (EGR1), forkhead box protein A3 (FOXA3), JUNB, Krüppel-like factor 9 (KLF9), KLF10, and REV-ERBα-were found to be up-regulated in liver during pregnancy. Results from transient transfection and promoter reporter gene assays indicate that KLF9 itself is a weak transactivator of CYP2D6 promoter but significantly enhances CYP2D6 promoter transactivation by hepatocyte nuclear factor 4 (HNF4α), a known transcriptional activator of CYP2D6 expression. The results from deletion and mutation analysis of CYP2D6 promoter activity identified a KLF9 putative binding motif at -22/-14 region to be critical in the potentiation of HNF4α-induced transactivation of CYP2D6. Electrophoretic mobility shift assays revealed a direct binding of KLF9 to the putative KLF binding motif. Results from chromatin immunoprecipitation assay showed increased recruitment of KLF9 to CYP2D6 promoter in the livers of tg-CYP2D6 mice during pregnancy. Taken together, our data suggest that increased KLF9 expression is in part responsible for CYP2D6 induction during pregnancy via the potentiation of HNF4α transactivation of CYP2D6.
Assuntos
Citocromo P-450 CYP2D6/genética , Fatores de Transcrição Kruppel-Like/fisiologia , Fígado/enzimologia , Prenhez/metabolismo , Animais , Feminino , Células HEK293 , Fator 4 Nuclear de Hepatócito/química , Fator 4 Nuclear de Hepatócito/fisiologia , Humanos , Fatores de Transcrição Kruppel-Like/química , Masculino , Camundongos , Gravidez , Regiões Promotoras GenéticasRESUMO
Renal tubular handling of urate is realized by a network of uptake and efflux transporters, including members of drug transporter families such as solute carrier proteins and ATP-binding cassette transporters. Solute carrier family 2, member 9 (SLC2A9), is one key factor of this so called "urate transportosome." The aim of the present study was to understand the transcriptional regulation of SLC2A9 and to test whether identified factors might contribute to a coordinated transcriptional regulation of the transporters involved in urate handling. In silico analysis and cell-based reporter gene assays identified a hepatocyte nuclear factor (HNF)4α-binding site in the promoter of SLC2A9 isoform 1, whose activity was enhanced by transient HNF4α overexpression, whereas mutation of the binding site diminished activation. HNF4α overexpression induced endogenous SLC2A9 expression in vitro. The in vivo role of HNF4α in the modulation of renal SLC2A9 gene expression was supported by findings of quantitative real-time RT-PCR analyses and chromatin immunoprecipitation assays. Indeed, mRNA expression of SLC2A9 and HNF4α in human kidney samples was significantly correlated. We also showed that in renal clear cell carcinoma, downregulation of HNF4α mRNA and protein expression was associated with a significant decline in expression of the transporter. Taken together, our data suggest that nuclear receptor family member HNF4α contributes to the transcriptional regulation of SLC2A9 isoform 1. Since HNF4α has previously been assumed to be a modulator of several urate transporters, our findings support the notion that there could be a transcriptional network providing synchronized regulation of the functional network of the urate transportosome.
Assuntos
Proteínas Facilitadoras de Transporte de Glucose/biossíntese , Fator 4 Nuclear de Hepatócito/fisiologia , Transportadores de Ânions Orgânicos/biossíntese , Sítios de Ligação/genética , Carcinoma de Células Renais/patologia , Carcinoma de Células Renais/fisiopatologia , Desdiferenciação Celular , Regulação da Expressão Gênica , Proteínas Facilitadoras de Transporte de Glucose/genética , Células HeLa , Humanos , Transportadores de Ânions Orgânicos/genética , Regiões Promotoras Genéticas/fisiologia , RNA Mensageiro/metabolismo , Transcrição GênicaRESUMO
BACKGROUND & AIMS: Hepatocyte-like cells, differentiated from different stem cell sources, are considered to have a range of possible therapeutic applications, including drug discovery, metabolic disease modelling, and cell transplantation. However, little is known about how stem cells differentiate into mature and functional hepatocytes. METHODS: Using transcriptomic screening, a transcription factor, liver X receptor α (NR1H3), was identified as increased during HepaRG cell hepatogenesis; this protein was also upregulated during embryonic stem cell and induced pluripotent stem cell differentiation. RESULTS: Overexpressing NR1H3 in human HepaRG cells promoted hepatic maturation; the hepatocyte-like cells exhibited various functions associated with mature hepatocytes, including cytochrome P450 (CYP) enzyme activity, secretion of urea and albumin, upregulation of hepatic-specific transcripts and an increase in glycogen storage. Importantly, the NR1H3-derived hepatocyte-like cells were able to rescue lethal fulminant hepatic failure using a non-obese diabetic/severe combined immunodeficient mouse model. CONCLUSIONS: In this study, we found that NR1H3 accelerates hepatic differentiation through an HNF4α-dependent reciprocal network. This contributes to hepatogenesis and is therapeutically beneficial to liver disease.
Assuntos
Diferenciação Celular/fisiologia , Fator 4 Nuclear de Hepatócito/fisiologia , Hepatócitos/fisiologia , Receptores Nucleares Órfãos/fisiologia , Células-Tronco/fisiologia , Animais , Tetracloreto de Carbono/efeitos adversos , Linhagem Celular , Transplante de Células , Modelos Animais de Doenças , Hepatócitos/citologia , Humanos , Técnicas In Vitro , Falência Hepática/induzido quimicamente , Falência Hepática/terapia , Regeneração Hepática/fisiologia , Receptores X do Fígado , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células-Tronco/citologiaRESUMO
The availability of pluripotent stem cells offers the possibility of using such cells to model hepatic disease and development. With this in mind, we previously established a protocol that facilitates the differentiation of both human embryonic stem cells and induced pluripotent stem cells into cells that share many characteristics with hepatocytes. The use of highly defined culture conditions and the avoidance of feeder cells or embryoid bodies allowed synchronous and reproducible differentiation to occur. The differentiation towards a hepatocyte-like fate appeared to recapitulate many of the developmental stages normally associated with the formation of hepatocytes in vivo. In the current study, we addressed the feasibility of using human pluripotent stem cells to probe the molecular mechanisms underlying human hepatocyte differentiation. We demonstrate (1) that human embryonic stem cells express a number of mRNAs that characterize each stage in the differentiation process, (2) that gene expression can be efficiently depleted throughout the differentiation time course using shRNAs expressed from lentiviruses and (3) that the nuclear hormone receptor HNF4A is essential for specification of human hepatic progenitor cells by establishing the expression of the network of transcription factors that controls the onset of hepatocyte cell fate.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Fator 4 Nuclear de Hepatócito/fisiologia , Hepatócitos/citologia , Fígado/embriologia , Células-Tronco Pluripotentes/citologia , Animais , Diferenciação Celular , Linhagem da Célula , Proliferação de Células , Fator 4 Nuclear de Hepatócito/metabolismo , Humanos , Lentivirus/genética , Camundongos , RNA Interferente Pequeno/metabolismoRESUMO
Hepatocyte nuclear factor 4 alpha (HNF4α), the master regulator of hepatocyte differentiation, has been recently shown to inhibit hepatocyte proliferation by way of unknown mechanisms. We investigated the mechanisms of HNF4α-induced inhibition of hepatocyte proliferation using a novel tamoxifen (TAM)-inducible, hepatocyte-specific HNF4α knockdown mouse model. Hepatocyte-specific deletion of HNF4α in adult mice resulted in increased hepatocyte proliferation, with a significant increase in liver-to-body-weight ratio. We determined global gene expression changes using Illumina HiSeq-based RNA sequencing, which revealed that a significant number of up-regulated genes following deletion of HNF4α were associated with cancer pathogenesis, cell cycle control, and cell proliferation. The pathway analysis further revealed that c-Myc-regulated gene expression network was highly activated following HNF4α deletion. To determine whether deletion of HNF4α affects cancer pathogenesis, HNF4α knockdown was induced in mice treated with the known hepatic carcinogen diethylnitrosamine (DEN). Deletion of HNF4α significantly increased the number and size of DEN-induced hepatic tumors. Pathological analysis revealed that tumors in HNF4α-deleted mice were well-differentiated hepatocellular carcinoma (HCC) and mixed HCC-cholangiocarcinoma. Analysis of tumors and surrounding normal liver tissue in DEN-treated HNF4α knockout mice showed significant induction in c-Myc expression. Taken together, deletion of HNF4α in adult hepatocytes results in increased hepatocyte proliferation and promotion of DEN-induced hepatic tumors secondary to aberrant c-Myc activation.
Assuntos
Carcinoma Hepatocelular/etiologia , Fator 4 Nuclear de Hepatócito/fisiologia , Hepatócitos/fisiologia , Neoplasias Hepáticas Experimentais/etiologia , Animais , Proliferação de Células , Dietilnitrosamina , Progressão da Doença , Deleção de Genes , Perfilação da Expressão Gênica , Homeostase , Masculino , Camundongos , Camundongos Knockout , Análise de Sequência de RNA , Tamoxifeno , TranscriptomaRESUMO
Vitamin A status regulates obesity development, hyperlipidemia, and hepatic lipogenic gene expression in Zucker fatty (ZF) rats. The development of hyperlipidemia in acne patients treated with retinoic acid (RA) has been attributed to the induction of apolipoprotein C-III expression. To understand the role of retinoids in the development of hyperlipidemia in ZF rats, the expression levels of several selected RA-responsive genes in the liver and isolated hepatocytes from Zucker lean (ZL) and ZF rats were compared using real-time PCR. The Rarb and Srebp-1c mRNA levels are higher in the liver and isolated hepatocytes from ZF than ZL rats. The Apoc3 mRNA level is only higher in the isolated hepatocytes from ZF than ZL rats. To determine whether dynamic RA production acutely regulates Apoc3 expression, its mRNA levels in response to retinoid treatments or adenovirus-mediated overexpression of hepatocyte nuclear factor 4 alpha (HNF4α) and chicken ovalbumin upstream-transcription factor II (COUP-TFII) were analyzed. Retinoid treatments for 2-6 h did not induce the expression of Apoc3 mRNA. The overexpression of HNF4α or COUP-TFII induced or inhibited Apoc3 expression, respectively. We conclude that short-term retinoid treatments could not induce Apoc3 mRNA expression, which is regulated by HNF4α and COUP-TFII in hepatocytes.