RESUMO
Acetaminophen (APAP) overdose is one of the leading causes of acute liver failure in the US and other developed countries, the molecular mechanisms of APAP-induced hepatotoxicity remain speculative. PIWI-interacting RNAs (piRNAs), a novel class of small non-coding RNAs, have been identified as epigenetic regulators of transposon silencing, mRNA deadenylation, and elimination. However, the functional role of piRNAs in APAP-induced liver injury remains unclear. In the current study, the piRNA profiles were constructed in HepaRG cells after APAP exposure, and the roles of piR-23210 in regulating nuclear receptors (NRs) expression, metabolizing enzymes expression, and consequently APAP-induced liver injury were systematically investigated. As a result, 57 upregulated piRNAs were identified after APAP exposure, indicating the stress-response characteristic of piRNA molecules. Subsequent in vitro and in vivo experiments proved that piR-23210 is a novel self-protective molecule that targets HNF1A and HNF4A transcripts by interacting with RNA binding protein Nucleolin (NCL), suppresses downstream CYPs (CYP2E1, CYP3A4, and CYP1A2) expression, and protects against APAP-induced liver injury. In conclusion, our findings provided new mechanistic clues revealing potential protective role of a piRNA against the hepatoxicity of APAP.
Assuntos
Acetaminofen/toxicidade , Analgésicos não Narcóticos/toxicidade , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Fator 4 Nuclear de Hepatócito/metabolismo , RNA Interferente Pequeno/metabolismo , Animais , Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Células HEK293 , Células Hep G2 , Fator 1-alfa Nuclear de Hepatócito/antagonistas & inibidores , Fator 4 Nuclear de Hepatócito/antagonistas & inibidores , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA Interferente Pequeno/administração & dosagemRESUMO
BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with poor prognosis, and gemcitabine-based chemotherapy remains an effective option for the majority of PDAC patients. Hepatocyte nuclear factor 1α (HNF1A) is a tumor-suppressor in PDAC, but its role in gemcitabine chemoresistance of PDAC has not been clarified. METHODS: The function of HNF1A in gemcitabine was detected by overexpression and knockdown of HNF1A in vitro and in vitro. The regulatory network between HNF1A and ABCB1 was further demonstrated by luciferase assays, deletion/mutation reporter construct assays and CHIP assays. FINDINGS: Here, we found that HNF1A expression is significantly associated with gemcitabine sensitivity in PDAC cell lines. Moreover, we identified that HNF1A overexpression enhanced gemcitabine sensitivity of PDAC both in vitro and in vitro, while inhibition of HNF1A had the opposite effect. Furthermore, by inhibiting and overexpressing HNF1A, we revealed that HNF1A regulates the expression of MDR genes (ABCB1 and ABCC1) in PDAC cells. Mechanistically, we demonstrated that HNF1A regulates ABCB1 expression through binding to its specific promoter region and suppressing its transcription levels. Finally, the survival analyses revealed the clinical value of HNF1A in stratification of gemcitabine sensitive pancreatic cancer patients. INTERPRETATION: Our study paved the road for finding novel treatment combinations using conventional cytotoxic agents with functional restoration of the HNF1A protein, individualized treatment through HNF1A staining and improvement of the prognosis of PDAC patients. FUND: National Natural Science Foundations of China and National Natural Science Foundation of Guangdong Province.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Desoxicitidina/análogos & derivados , Resistencia a Medicamentos Antineoplásicos , Fator 1-alfa Nuclear de Hepatócito/antagonistas & inibidores , Neoplasias Pancreáticas/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/antagonistas & inibidores , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Adulto , Idoso , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células , Desoxicitidina/farmacologia , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Expressão Gênica , Fator 1-alfa Nuclear de Hepatócito/genética , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Pessoa de Meia-Idade , Gradação de Tumores , Metástase Neoplásica , Estadiamento de Neoplasias , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/mortalidade , Neoplasias Pancreáticas/patologia , Prognóstico , Regiões Promotoras Genéticas , Ligação Proteica , GencitabinaRESUMO
Under diabetic conditions, sodium-glucose cotransporter 2 (SGLT2) for glucose uptake in proximal tubules (PTs) increases, whereas NAD+-dependent protein deacetylase silent mating type information regulation 2 homolog 1 (Sirtuin-1; SIRT1) for PT survival decreases. Therefore, we hypothesized that increased glucose influx by SGLT2 reduces SIRT1 expression. To test this hypothesis, db/db mice with diabetes and high-glucose (HG)-cultured porcine PT LLC-PK1 cells in a two-chamber system were treated with the SGLT2 inhibitor canagliflozin. We also examined SIRT1 and SGLT2 expression in human kidney biopsies. In db/db mice, SGLT2 expression increased with concomitant decreases in SIRT1, but was inhibited by canagliflozin. For determination of the polarity of SGLT2 and SIRT1 expression, LLC-PK1 cells were seeded into Transwell chambers (pore size, 0.4 µm; Becton Dickinson, Oxford, UK). HG medium was added to either or to both of the upper and lower chambers, which corresponded to the apical and basolateral sides of the cells, respectively. In this system, the lower chamber with HG showed increased SGLT2 and decreased SIRT1 expression. Canagliflozin reversed HG-induced SIRT1 downregulation. Gene silencing and inhibitors for glucose transporter 2 (GLUT2) blocked HG-induced SGLT2 expression upregulation. Gene silencing for the hepatic nuclear factor-1α (HNF-1α), whose nuclear translocation was enhanced by HG, blocked HG-induced SGLT2 expression upregulation. Similarly, gene silencing for importin-α1, a chaperone protein bound to GLUT2, blocked HG-induced HNF-1α nuclear translocation and SGLT2 expression upregulation. In human kidney, SIRT1 immunostaining was negatively correlated with SGLT2 immunostaining. Thus, under diabetic conditions, SIRT1 expression in PTs was downregulated by an increase in SGLT2 expression, which was stimulated by basolateral HG through activation of the GLUT2/importin-α1/HNF-1α pathway.
Assuntos
Diabetes Mellitus Experimental/genética , Nefropatias Diabéticas/genética , Transportador de Glucose Tipo 2/genética , Glucose/farmacologia , Sirtuína 1/genética , Transportador 2 de Glucose-Sódio/genética , Animais , Canagliflozina/farmacologia , Linhagem Celular , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Cultura em Câmaras de Difusão , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Regulação da Expressão Gênica , Transportador de Glucose Tipo 2/antagonistas & inibidores , Transportador de Glucose Tipo 2/metabolismo , Fator 1-alfa Nuclear de Hepatócito/antagonistas & inibidores , Fator 1-alfa Nuclear de Hepatócito/genética , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Humanos , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Sirtuína 1/metabolismo , Transportador 2 de Glucose-Sódio/metabolismo , Inibidores do Transportador 2 de Sódio-Glicose/farmacologia , Suínos , alfa Carioferinas/antagonistas & inibidores , alfa Carioferinas/genética , alfa Carioferinas/metabolismoRESUMO
6-Formylindolo(3,2-b)carbazole (FICZ), a high-affinity aryl hydrocarbon receptor (AhR) ligand, plays a protective role in inflammatory bowel disease (IBD) through activation of AhR. Interleukin-6 (IL-6) induced intestinal epithelial barrier dysfunction is involved in the pathological process of IBD. In this study, we investigated the protective effects of FICZ on IL-6 induced intestinal epithelial barrier injury. Our data show that AhR activation by FICZ ameliorated colonic inflammation, decreased IL-6 and claudin-2 expression, and maintained intestinal barrier function in a mouse model of dextran sulphate sodium (DSS)-induced colitis. In Caco-2 and T84 intestinal epithelial cells, FICZ also prevented the increase of intestinal epithelial permeability and claudin-2 expression induced by IL-6. Depletion of AhR expression by small interfering (si)RNA reversed FICZ induced decrease of claudin-2. Furthermore, IL-6 induced upregulation of claudin-2 was required for increased caudal-related homeobox 2 (CDX-2) and hepatocyte-nuclear factor (HNF)-1α. However, FICZ repressed the increase of CDX-2 and HNF-1α expression induced by IL-6. These results reveal the protective effects of FICZ on IL-6 induced disruption of intestinal epithelial barrier function through suppressing the expression of claudin-2. In addition, CDX-2 and HNF-1α are involved in the regulation of claudin-2 after IL-6 and FICZ treatment. Therefore compounds related to AhR ligands may be potential pharmaceutical agents to treat IBD.
Assuntos
Carbazóis/farmacologia , Claudina-2/metabolismo , Regulação para Baixo/efeitos dos fármacos , Receptores de Hidrocarboneto Arílico/metabolismo , Animais , Fator de Transcrição CDX2/antagonistas & inibidores , Fator de Transcrição CDX2/genética , Fator de Transcrição CDX2/metabolismo , Células CACO-2 , Carbazóis/química , Claudina-2/genética , Colite/induzido quimicamente , Colite/patologia , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Sulfato de Dextrana/toxicidade , Modelos Animais de Doenças , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Fator 1-alfa Nuclear de Hepatócito/antagonistas & inibidores , Fator 1-alfa Nuclear de Hepatócito/genética , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Humanos , Interleucina-6/farmacologia , Intestinos/citologia , Intestinos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Hidrocarboneto Arílico/antagonistas & inibidores , Receptores de Hidrocarboneto Arílico/genética , Regulação para Cima/efeitos dos fármacosRESUMO
Chitosan oligosaccharides (COS), linear polymers of N-acetyl-D-glucosamine and deacetylated glucosamine, exhibit diverse pharmacological effects such as antimicrobial, antitumor, antioxidant and anti-inflammatory activities. Here, we explored their hypocholesterolemic effects in vivo and the molecular mechanisms of COS in hepatic cells. Our in vivo study of dyslipidemic ApoE-/- male mice showed that COS treatment of 500â¯mgâ¯kg-1 d-1 for 4 weeks clearly reduced the lipid deposits in the aorta and significantly decreased the hepatic proprotein convertase subtilisin/kexin type 9 (PCSK9) protein levels versus HFD groups (pâ¯<â¯0.05). To elucidate the mechanisms behind these effects, the HepG2 cell line was treated with COS. We found that COS (200⯵g/ml) increased the amount of cell-surface low-density lipoprotein receptor (LDLR) and enhanced the lipid droplets in HepG2 cells (pâ¯<â¯0.05). The mRNA levels of LDLR and HMG-CoA protein levels were not altered, and the mRNA levels of PCSK9 were down-regulated by COS treatment for 24â¯h. We also observed that the expression levels of SREBP-2 (125 kD) and HNF-1α were increased in total cell lysates, but nuclear SREBP-2 (nSREBP-2, 68 kD, the active subunit of SREBP-2) levels were decreased and FOXO3a levels increased in nuclear lysates after COS treatment for 24â¯h. We demonstrated that one of the reasons for regulation of lipid transfer with COS is that FOXO3a levels are up-regulated by COS, leading to a reduction in the PCSK9 promoter binding capacity of HNF-1α and thus suppressing PCSK9 gene expression, up-regulating LDLR levels, and enhancing the lipid droplets in HepG2 cells. In addition, decreased expression of the PCSK9 gene was also contributed to by down-regulation of SREBP-2 by COS. We further confirmed the effect of suppression of PCSK9 expression by COS by utilizing RNA interference to silence HNF-1α and SREBP-2. Finally, to the best of our knowledge, we are the first to demonstrate that PCSK9 expression and LDLR activity are synergistically changed by a combination of HNF-1α and SREBP-2 after COS treatment. Our findings indicate that COS may regulate PCSK9 to modulate hepatic LDLR abundance and activity.
Assuntos
Aterosclerose/prevenção & controle , Carcinoma Hepatocelular/prevenção & controle , Quitosana/farmacologia , Gotículas Lipídicas/metabolismo , Oligossacarídeos/farmacologia , Pró-Proteína Convertase 9/metabolismo , Pró-Proteína Convertase 9/fisiologia , Animais , Anticolesterolemiantes/farmacologia , Apolipoproteínas E/fisiologia , Aterosclerose/metabolismo , Aterosclerose/patologia , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Células Cultivadas , Regulação para Baixo , Fator 1-alfa Nuclear de Hepatócito/antagonistas & inibidores , Fator 1-alfa Nuclear de Hepatócito/genética , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/prevenção & controle , Masculino , Camundongos , Inibidores de PCSK9 , Pró-Proteína Convertase 9/genética , RNA Interferente Pequeno/genética , Receptores de LDL/genética , Receptores de LDL/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 2/antagonistas & inibidores , Proteína de Ligação a Elemento Regulador de Esterol 2/genética , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismoRESUMO
Continuous lifetime exposure to certain natural and man-made chemicals is a major cause of cancers in humans; therefore, evaluating the carcinogenic risks of chemicals remains important. Currently, substantial progress has been made in identification of genotoxic carcinogens; in contrast, predicting the carcinogenic potential of nongenotoxic compounds is a challenge due to many different modes of action that may lead to tumorigenesis. In the present study, we investigated the effects of the nongenotoxic liver carcinogen methapyrilene and the nongenotoxic noncarcinogen usnic acid, at doses that do not exhibit organ cytotoxicity, on epigenomic alterations in the livers and kidneys of Fischer 344 (F344) rats. We demonstrate that a repeat-dose oral treatment of male F344 rats with methapyrilene for 6 weeks caused target organ-specific epigenetic alterations in the livers. In contrast, only very slight epigenetic changes were found in the livers of F344 rats treated with hepatotoxicant, but noncarcinogen, usnic acid. The methapyrilene-induced epigenetic changes consisted of changes in histone lysine acetylation and methylation, with the greatest increase occurring in global and gene-specific histone H3 lysine 9 (H3K9) deacetylation. Importantly, the results of the present study show an association between gene-specific histone H3K9 deacetylation and a reduced expression of critical cancer-related genes, including prospero homeobox 1 (Prox1), HNF1 homebox A (Hnf1a), and peroxisome proliferator activated receptor alpha (Ppara), which provides a mechanistic link between methapyrilene-induced epigenetic aberrations and liver carcinogenesis.
Assuntos
Carcinógenos/toxicidade , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Epigênese Genética/efeitos dos fármacos , Histonas/metabolismo , Fígado/efeitos dos fármacos , Metapirileno/toxicidade , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Acetilação/efeitos dos fármacos , Administração Oral , Animais , Benzofuranos/administração & dosagem , Benzofuranos/toxicidade , Carcinógenos/administração & dosagem , Relação Dose-Resposta a Droga , Fator 1-alfa Nuclear de Hepatócito/antagonistas & inibidores , Fator 1-alfa Nuclear de Hepatócito/genética , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Proteínas de Homeodomínio/antagonistas & inibidores , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Rim/efeitos dos fármacos , Rim/metabolismo , Fígado/metabolismo , Lisina/metabolismo , Masculino , Metapirileno/administração & dosagem , Metilação/efeitos dos fármacos , Especificidade de Órgãos , PPAR alfa/antagonistas & inibidores , PPAR alfa/genética , PPAR alfa/metabolismo , Distribuição Aleatória , Ratos Endogâmicos F344 , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
BACKGROUND: Increasing evidence has demonstrated that long non-coding RNAs (lncRNAs) play essential roles in the occurrence and development of human cancers, including gastric cancer (GC). However, the functional and clinical significance of lncRNAs are still poorly understood. METHODS: In this study, the expression of LncRNA HNF1A antisense RNA 1 (HNF1A-AS1) was first examined by lncRNAs microarray analysis in 6 GC tissues, and was then further verified by real-time quantitative reverse transcription PCR (qRT-PCR) both in 3 GC cell lines and 161 cases of GC tissues. We also evaluated the association between HNF1A-AS1 expression and clinicopathological features of patients with GC. RESULTS: LncRNAs microarray analysis results exhibited that HNF1A-AS1 was downregulated in GCs tissues (mean fold change 2.06, p < 0.05), which was further confirmed by qRT-PCR. The results from qRT-PCR showed that the expression of HNF1A-AS1 was not only downregulated in three GC cell lines (AGS, BGC-823, and MKN-45) relative to that in a normal gastric mucosal epithelial cell line (GES-1), but also decreased in GC tissues relative to that in paired adjacent non-neoplastic tissues (low expression, 94 of 161; low expression rate, 58.38%). Furthermore, low HNF1A-AS1 expression was associated with tumor size/diameter (p = 0.005, multivariate analysis), levels of serum carcinoembryonic antigen (CEA), and carbohydrate antigen 19-9 (CA19-9), and RRM1 expression in tissue samples (p = 0.028, p = 0.009, and p = 0.006, respectively). CONCLUSIONS: Taken together, our data indicate that lncRNA HNF1A-AS1 may be a regulator of GC, and thus, it may have potential as a novel biomarker and treatment target for this type of cancer.
Assuntos
Adenocarcinoma/genética , Mucosa Gástrica/metabolismo , Fator 1-alfa Nuclear de Hepatócito/antagonistas & inibidores , RNA Longo não Codificante/genética , RNA Interferente Pequeno/genética , Neoplasias Gástricas/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/secundário , Proliferação de Células , Células Cultivadas , Feminino , Seguimentos , Fator 1-alfa Nuclear de Hepatócito/genética , Humanos , Técnicas Imunoenzimáticas , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Estadiamento de Neoplasias , Prognóstico , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologiaRESUMO
Our previous in vitro studies have identified hepatocyte nuclear factor 1α (HNF1α) as an obligated trans-activator for PCSK9 gene expression and demonstrated its functional involvement in the suppression of PCSK9 expression by berberine (BBR), a natural cholesterol-lowering compound. In this study, we investigated the mechanism underlying the inhibitory effect of BBR on HNF1α-mediated PCSK9 transcription. Administration of BBR to hyperlipidemic mice and hamsters lowered circulating PCSK9 concentrations and hepatic PCSK9 mRNA levels without affecting the gene expression of HNF1α. However, hepatic HNF1α protein levels were markedly reduced in BBR-treated animals as compared with the control. Using HepG2 cells as a model system, we obtained evidence that BBR treatment let to accelerated degradation of HNF1α protein. By applying inhibitors to selectively block the ubiquitin proteasome system (UPS) and autophagy-lysosomal pathway, we show that HNF1α protein content in HepG2 cells was not affected by bafilomycin A1 treatment, but it was dose-dependently increased by UPS inhibitors bortezomib and MG132. Bortezomib treatment elevated HNF1α and PCSK9 cellular levels with concomitant reductions of LDL receptor protein. Moreover, HNF1α protein displayed a multiubiquitination ladder pattern in cells treated with BBR or overexpressing ubiquitin. By expressing GFP-HNF1α fusion protein in cells, we observed that blocking UPS resulted in accumulation of GFP-HNF1α in cytoplasm. Importantly, we show that the BBR reducing effects on HNF1α protein and PCSK9 gene transcription can be eradicated by proteasome inhibitors. Altogether, our studies using BBR as a probe uncovered a new aspect of PCSK9 regulation by ubiquitin-induced proteasomal degradation of HNF1α.
Assuntos
Berberina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Fígado/efeitos dos fármacos , Pró-Proteína Convertases/antagonistas & inibidores , Pró-Proteína Convertases/genética , Serina Endopeptidases/genética , Ubiquitina/metabolismo , Animais , Western Blotting , Células Cultivadas , Cricetinae , Células HEK293 , Células Hep G2 , Fator 1-alfa Nuclear de Hepatócito/antagonistas & inibidores , Fator 1-alfa Nuclear de Hepatócito/genética , Humanos , Fígado/metabolismo , Masculino , Mesocricetus , Camundongos , Regiões Promotoras Genéticas/genética , Pró-Proteína Convertase 9 , Pró-Proteína Convertases/metabolismo , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Serina Endopeptidases/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
OBJECTIVE: Pitavastatin, a highly potent inhibitor of 3-hydroxy-methylglutarylcoenzyme A reductase, is a known substrate of OATP1B1. Ursodeoxycholic acid (UDCA) inhibits OATP1B1 expression by repressing hepatocyte nuclear factor 1α (HNF1α). Thus, the effects of UDCA on the pharmacokinetics of pitavastatin were investigated in healthy subjects. METHODS: An open-label, 2-phase, parallel study was conducted with 13 healthy volunteers. In the control phase, after an overnight fast, each subject received a single dose of 2 mg pitavastatin. After a 1-week washout period, in the UDCA phase, subjects received a daily oral dose of 600 mg of UDCA (300 mg b.i.d.) for 14 days. On day 15, 2 mg of pitavastatin was administered as described previously for the control phase. RESULTS: In the UDCA phase, the maximum plasma concentration (C(max)) of pitavastatin was slightly higher than in the control phase (48.6 ± 22.9 ng/mL vs. 42.4 ± 16.1 ng/mL). However, the overall pharmacokinetic parameters of pitavastatin and pitavastatin lactone during the two study phases were not significantly different. CONCLUSIONS: UDCA had no significant effect on the pharmacokinetics of pitavastatin. These results do not support the notion that UDCA increases the systemic exposure of OATP1B1 substrate by inhibiting HNF1α and decreasing OATP1B1 transporter expression.
Assuntos
Fator 1-alfa Nuclear de Hepatócito/antagonistas & inibidores , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacocinética , Quinolinas/farmacocinética , Ácido Ursodesoxicólico/farmacologia , Interações Medicamentosas , Humanos , Transportador 1 de Ânion Orgânico Específico do Fígado , Masculino , Transportadores de Ânions Orgânicos/fisiologiaRESUMO
Cyclic activation of the Wnt/ß-catenin signaling pathway controls cell fusion-mediated somatic cell reprogramming. TCFs belong to a family of transcription factors that, in complex with ß-catenin, bind and transcriptionally regulate Wnt target genes. Here, we show that Wnt/ß-catenin signaling needs to be off during the early reprogramming phases of mouse embryonic fibroblasts (MEFs) into iPSCs. In MEFs undergoing reprogramming, senescence genes are repressed and mesenchymal-to-epithelial transition is favored. This is correlated with a repressive activity of TCF1, which contributes to the silencing of Wnt/ß-catenin signaling at the onset of reprogramming. In contrast, the Wnt pathway needs to be active in the late reprogramming phases to achieve successful reprogramming. In conclusion, continued activation or inhibition of the Wnt/ß-catenin signaling pathway is detrimental to the reprogramming of MEFs; instead, temporal perturbation of the pathway is essential for efficient reprogramming, and the "Wnt-off" state can be considered an early reprogramming marker.
Assuntos
Fator 1-alfa Nuclear de Hepatócito/metabolismo , Via de Sinalização Wnt , Animais , Antibióticos Antineoplásicos/farmacologia , Linhagem Celular , Reprogramação Celular/efeitos dos fármacos , Doxorrubicina/farmacologia , Transição Epitelial-Mesenquimal , Fator 1-alfa Nuclear de Hepatócito/antagonistas & inibidores , Fator 1-alfa Nuclear de Hepatócito/genética , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , beta Catenina/genética , beta Catenina/metabolismoRESUMO
A critical cis-regulatory element for the CFTR (cystic fibrosis transmembrane conductance regulator) gene is located in intron 11, 100 kb distal to the promoter, with which it interacts. This sequence contains an intestine-selective enhancer and associates with enhancer signature proteins, such as p300, in addition to tissue-specific TFs (transcription factors). In the present study we identify critical TFs that are recruited to this element and demonstrate their importance in regulating CFTR expression. In vitro DNase I footprinting and EMSAs (electrophoretic mobility-shift assays) identified four cell-type-selective regions that bound TFs in vitro. ChIP (chromatin immunoprecipitation) identified FOXA1/A2 (forkhead box A1/A2), HNF1 (hepatocyte nuclear factor 1) and CDX2 (caudal-type homeobox 2) as in vivo trans-interacting factors. Mutation of their binding sites in the intron 11 core compromised its enhancer activity when measured by reporter gene assay. Moreover, siRNA (small interfering RNA)-mediated knockdown of CDX2 caused a significant reduction in endogenous CFTR transcription in intestinal cells, suggesting that this factor is critical for the maintenance of high levels of CFTR expression in these cells. The ChIP data also demonstrate that these TFs interact with multiple cis-regulatory elements across the CFTR locus, implicating a more global role in intestinal expression of the gene.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Elementos Facilitadores Genéticos , Enterócitos/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Proteínas de Homeodomínio/metabolismo , Transativadores/metabolismo , Fator de Transcrição CDX2 , Linhagem Celular , Regulador de Condutância Transmembrana em Fibrose Cística/química , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fatores de Transcrição Forkhead/antagonistas & inibidores , Fatores de Transcrição Forkhead/química , Fatores de Transcrição Forkhead/genética , Regulação da Expressão Gênica , Genes Reporter , Fator 1-alfa Nuclear de Hepatócito/antagonistas & inibidores , Fator 1-alfa Nuclear de Hepatócito/química , Fator 1-alfa Nuclear de Hepatócito/genética , Proteínas de Homeodomínio/antagonistas & inibidores , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/genética , Humanos , Íntrons , Proteínas Mutantes/antagonistas & inibidores , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Interferência de RNA , RNA Mensageiro/metabolismo , RNA Interferente Pequeno , Proteínas Recombinantes de Fusão/antagonistas & inibidores , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Transativadores/antagonistas & inibidores , Transativadores/química , Transativadores/genéticaRESUMO
Apolipoprotein M (ApoM) is a recently discovered human apolipoprotein predominantly present in high-density lipoprotein (HDL) in the plasma. Statins have effects on many HDL-associated apolipoproteins. However, it is unknown whether statins have effects on ApoM. In the present study, we investigated the effects of simvastatin on ApoM expression and the underlying mechanism(s). Simvastatin up-regulated hepatic ApoM mRNA and protein expression in mice. In HepG2 cells, simvastatin significantly enhanced ApoM mRNA and protein expression in a dose-dependent manner. Simvastatin increased hepatic hepatocyte nuclear factor-1α (HNF-1α) mRNA and reduced liver X receptor-α (LXRα) mRNA expression in mice. The simvastatin-induced up-regulation of ApoM was blocked by an HNF-1α inhibitor (UCDA) or an LXRα agonist (TO901317) in HepG2 cells which indicates that this effect is mediated via the regulation of HNF-1α and LXRα. In conclusion, simvastatin signiï¬cantly up-regulated ApoM expression in vivo and in vitro, which indicates that ApoM is another novel apolipoprotein regulated by simvastatin. The mechanism of this effect is related to the regulation of HNF-1α and LXRα.
Assuntos
Anticolesterolemiantes/farmacologia , Apolipoproteínas/genética , Apolipoproteínas/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Sinvastatina/farmacologia , Animais , Células Hep G2 , Fator 1-alfa Nuclear de Hepatócito/antagonistas & inibidores , Fator 1-alfa Nuclear de Hepatócito/genética , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Receptores X do Fígado , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptores Nucleares Órfãos/agonistas , Receptores Nucleares Órfãos/genética , Receptores Nucleares Órfãos/metabolismo , RNA Mensageiro/metabolismoRESUMO
BACKGROUND: Hepatocyte Nuclear Factor 1α (HNF1α) is an atypical homeodomain-containing transcription factor that transactivates liver-specific genes including albumin, α-1-antitrypsin and α- and ß-fibrinogen. Biallelic inactivating mutations of HNF1A have been frequently identified in hepatocellular adenomas (HCA), rare benign liver tumors usually developed in women under oral contraceptives, and in rare cases of hepatocellular carcinomas developed in non-cirrhotic liver. HNF1α-mutated HCA (H-HCA) are characterized by a marked steatosis and show activation of glycolysis, lipogenesis, translational machinery and mTOR pathway. We studied the consequences of HNF1α silencing in hepatic cell lines, HepG2 and Hep3B and we reproduced most of the deregulations identified in H-HCA. METHODS: We transfected hepatoma cell lines HepG2 and Hep3B with siRNA targeting HNF1α and obtained a strong inhibition of HNF1α expression. We then looked at the phenotypic changes by microscopy and studied changes in gene expression using qRT-PCR and Western Blot. RESULTS: Hepatocytes transfected with HNF1α siRNA underwent severe phenotypic changes with loss of cell-cell contacts and development of migration structures. In HNF1α-inhibited cells, hepatocyte and epithelial markers were diminished and mesenchymal markers were over-expressed. This epithelial-mesenchymal transition (EMT) was related to the up regulation of several EMT transcription factors, in particular SNAIL and SLUG. We also found an overexpression of TGFß1, an EMT initiator, in both cells transfected with HNF1α siRNA and H-HCA. Moreover, TGFß1 expression is strongly correlated to HNF1α expression in cell models, suggesting regulation of TGFß1 expression by HNF1α. CONCLUSION: Our results suggest that HNF1α is not only important for hepatocyte differentiation, but has also a role in the maintenance of epithelial phenotype in hepatocytes.
Assuntos
Transição Epitelial-Mesenquimal/genética , Fator 1-alfa Nuclear de Hepatócito/antagonistas & inibidores , Neoplasias Hepáticas/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Células Epiteliais/metabolismo , Expressão Gênica , Inativação Gênica , Células Hep G2 , Fator 1-alfa Nuclear de Hepatócito/genética , Humanos , Mutação , Fenótipo , Transfecção , Fator de Crescimento Transformador beta1/genéticaRESUMO
UNLABELLED: With about 350 million virus carriers, hepatitis B virus (HBV) infection remains a major health problem. HBV is a noncytopathic virus causing persistent infection, but it is still unknown whether host recognition of HBV may activate an innate immune response. We describe that upon infection of primary human liver cells, HBV is recognized by nonparenchymal cells of the liver, mainly by liver macrophages (Kupffer cells), although they are not infected. Within 3 hours, this recognition leads to the activation of nuclear factor kappa B (NF-kappaB) and subsequently to the release of interleukin-6 (IL-6) and other proinflammatory cytokines (IL-8, TNF-alpha, IL-1beta), but does not induce an interferon response. The activation of proinflammatory cytokines, however, is transient, and even inhibits responsiveness toward a subsequent challenge. IL-6 released by Kupffer cells after activation of NF-kappaB controls HBV gene expression and replication in hepatocytes at the level of transcription shortly after infection. Upon binding to its receptor complex, IL-6 activates the mitogen-activated protein kinases exogenous signal-regulated kinase 1/2, and c-jun N-terminal kinase, which inhibit expression of hepatocyte nuclear factor (HNF) 1alpha and HNF 4alpha, two transcription factors essential for HBV gene expression and replication. CONCLUSION: Our results demonstrate recognition of HBV patterns by nonparenchymal liver cells, which results in IL-6-mediated control of HBV infection at the transcriptional level. Thus, IL-6 ensures early control of the virus, limiting activation of the adaptive immune response and preventing death of the HBV-infected hepatocyte. This pattern recognition may be essential for a virus, which infects a new host with only a few virions. Our data also indicate that therapeutic neutralization of IL-6 for treatment of certain diseases may represent a risk if the patient is HBV-infected.
Assuntos
Regulação da Expressão Gênica , Hepatite B/imunologia , Interferons/fisiologia , Interleucina-6/fisiologia , Células Cultivadas , Citocinas/biossíntese , Hepatite B/metabolismo , Vírus da Hepatite B/efeitos dos fármacos , Vírus da Hepatite B/genética , Vírus da Hepatite B/fisiologia , Fator 1-alfa Nuclear de Hepatócito/antagonistas & inibidores , Fator 1-alfa Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/antagonistas & inibidores , Fator 4 Nuclear de Hepatócito/genética , Hepatócitos/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases , NF-kappa B/metabolismo , Transcrição Gênica , Replicação ViralRESUMO
Expression of the organic anion transporting polypeptide 1B1 (OATP1B1) is regulated by transcription factor hepatic nuclear factor (HNF) 1alpha. The aim of this study was to investigate the effect of ursodeoxycholic acid (UDCA), an inhibitor of transcription factor HNF1alpha, on rosuvastatin and bilirubin kinetics in human healthy volunteers. Both substances are substrates of OATP1B1. Twelve subjects with OATP1B1(*)1b/(*)1b genotype predicting high transport activity were recruited for this randomized, crossover study. Each subject received a single p.o. dose of 20 mg of rosuvastatin after 14 days of p.o. intake of either 500 mg of UDCA or placebo. Plasma concentrations of rosuvastatin were determined on days 15 to 18 of each study period. Subjects were randomly assigned to UDCA or placebo group. Intake of UDCA led to a significant increase in rosuvastatin area under the curve (AUC)(0-72) from 128.5 ng/ml.h to 182.1 ng/ml.h(P = 0.008) compared with the control group. The oral clearance decreased from 155.2 l/h with placebo to 109.8 l/h with UDCA. In addition, the mean values of total bilirubin, conjugated bilirubin, and unconjugated bilirubin significantly increased to 139 +/- 39% (P = 0.003), 127 +/- 29% (P = 0.005), and 151 +/- 52% (P = 0.004), respectively, after UDCA treatment. These results in healthy volunteers confirm the findings from in vitro studies that UDCA inhibits OATP1B1 activity by inhibition of the transcription factor HNF1alpha. They highlight a novel mechanism of OATP1B1-based interaction that is mediated by transcription factor HNF1alpha.
Assuntos
Bilirrubina/sangue , Fluorbenzenos/farmacocinética , Fator 1-alfa Nuclear de Hepatócito/antagonistas & inibidores , Transportadores de Ânions Orgânicos/metabolismo , Pirimidinas/farmacocinética , Sulfonamidas/farmacocinética , Ácido Ursodesoxicólico/farmacologia , Adulto , Área Sob a Curva , Estudos Cross-Over , Método Duplo-Cego , Fluorbenzenos/metabolismo , Humanos , Masculino , Estudos Prospectivos , Pirimidinas/metabolismo , Rosuvastatina Cálcica , Sulfonamidas/metabolismoRESUMO
Normal cellular models able to efficiently recapitulate intestinal epithelial cell differentiation in culture are not yet available. The aim of this work was to establish and genetically characterize a mesenchymal-epithelial coculture system to identify transcriptional regulators involved in this process. The deposition of rat intestinal epithelial cells on human intestinal mesenchymal cells led to the formation of clustered structures that expanded shortly after seeding. These structures were composed of polarized epithelial cells with brush borders and cell junction complexes. A rat GeneChip statistical analysis performed at different time points during this process identified hepatocyte nuclear factor-4alpha (HNF-4alpha) and hepatocyte nuclear factor-1alpha (HNF-1alpha) as being induced coincidently with the apparition of polarized epithelial structures. Stable introduction of HNF-4alpha in undifferentiated epithelial cells alone led to the rapid induction of HNF-1alpha and several intestinal-specific markers and metabolism-related genes for which mRNA was identified to be upregulated during epithelial differentiation. HNF-4alpha was capable to transactivate the calbindin 3 gene promoter, a process that was synergistically increased in the presence of HNF-1alpha. When HNF-4alpha-expressing cells were plated on mesenchymal cells, an epithelial monolayer formed rapidly with the apparition of dome structures that are characteristics of vectorial ion transport. Forced expression of HNF-1alpha alone did not result in dome structures formation. In sum, this novel coculture system functionally identified for the first time HNF-4alpha as an important modulator of intestinal epithelial differentiation and offers an innovative opportunity to investigate molecular mechanisms involved in this process.
Assuntos
Células Epiteliais/efeitos dos fármacos , Fator 4 Nuclear de Hepatócito/farmacologia , Intestinos/citologia , Adulto , Western Blotting , Fator de Transcrição CDX2 , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Técnicas de Cocultura , Interpretação Estatística de Dados , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Perfilação da Expressão Gênica , Marcadores Genéticos , Fator 1-alfa Nuclear de Hepatócito/antagonistas & inibidores , Proteínas de Homeodomínio/fisiologia , Humanos , Intestinos/efeitos dos fármacos , Microscopia Eletrônica , Análise de Sequência com Séries de Oligonucleotídeos , Gravidez , RNA Interferente Pequeno , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Aberrant accumulation of beta-catenin is closely related to carcinogenesis. Mutations in the p53 gene are reported to induce the aberrant accumulation of beta-catenin in the absence of dysfunction in the glycogen synthase kinase 3beta (GSK3beta)-mediated degradation pathway, but the mechanism remains incompletely understood. Here, we show that human coiled-coil domain containing 85B (CCDC85B) is induced by p53 and regulates beta-catenin activity via interaction with the T-cell factor 4 in the nucleus. Moreover, CCDC85B enhances the degradation of beta-catenin and suppresses tumor cell growth. In conclusion, we revealed that CCDC85B-induced degradation of beta-catenin is independent of GSK3beta and other p53-inducible products, Siah-1L, suggesting that CCDC85B constitutes the one of the frameworks of p53-induced multiple regulatory pathways for beta-catenin activity.
Assuntos
Proteínas de Transporte/fisiologia , Proteína Supressora de Tumor p53/metabolismo , beta Catenina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Núcleo Celular/metabolismo , Células Cultivadas , Inibidores de Cisteína Proteinase/farmacologia , Imunofluorescência , Perfilação da Expressão Gênica , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Fator 1-alfa Nuclear de Hepatócito/antagonistas & inibidores , Fator 1-alfa Nuclear de Hepatócito/genética , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Humanos , Immunoblotting , Imunoprecipitação , Leupeptinas/farmacologia , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Repressoras , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição TCF/genética , Fatores de Transcrição TCF/metabolismo , Proteína 2 Semelhante ao Fator 7 de Transcrição , Transfecção , Ubiquitina/metabolismoRESUMO
Biallelic inactivating mutations of the transcription factor 1 gene (TCF1), encoding hepatocyte nuclear factor 1alpha (HNF1alpha) were identified in 50% of hepatocellular adenomas (HCA) phenotypically characterized by a striking steatosis. To understand the molecular basis of this aberrant lipid storage, we performed a microarray transcriptome analysis validated by quantitative reverse transcription-PCR, Western blotting, and lipid profiling. In mutated HCA, we showed a repression of gluconeogenesis coordinated with an activation of glycolysis, citrate shuttle, and fatty acid synthesis predicting elevated rates of lipogenesis. Moreover, the strong down-regulation of liver fatty acid-binding protein suggests that impaired fatty acid trafficking may also contribute to the fatty phenotype. In addition, transcriptional profile analysis of the observed deregulated genes in non-HNF1alpha-mutated HCA as well as in non-tumor livers allowed us to define a specific signature of the HNF1alpha-mutated HCA. In these tumors, lipid composition was dramatically modified according to the transcriptional deregulations identified in the fatty acid synthetic pathway. Surprisingly, lipogenesis activation did not operate through sterol regulatory element-binding protein-1 (SREBP-1) and carbohydrate-response element-binding protein (ChREBP) that were repressed. We conclude that steatosis in HNF1alpha-mutated HCA results mainly from an aberrant promotion of lipogenesis that is linked to HNF1alpha inactivation and that is independent of both SREBP-1 and ChREBP activation. Finally, our findings have potential clinical implications since lipogenesis can be efficiently inhibited by targeted therapies.