RESUMEN
Pancreatic cancer (PC) is difficult to defeat due to mechanism (s) driving metastasis and drug resistance. Cancer stemness is a major challenging phenomenon associated with PC metastasis and limiting therapy efficacy. In this study, we evaluated the pre-clinical and clinical significance of eradicating pancreatic cancer stem cells (PCSC) and its components using a pan-EGFR inhibitor afatinib in combination with gemcitabine. Afatinib in combination with gemcitabine significantly reduced KrasG12D/+; Pdx-1 Cre (KC) (P < 0.01) and KrasG12D/+; p53R172H/+; Pdx-1 Cre (KPC) (P < 0.05) derived mouse tumoroids and KPC-derived murine syngeneic cell line growth compared to gemcitabine/afatinib alone treatment. The drug combination also reduced PC xenograft tumor burden (P < 0.05) and the incidence of metastasis by affecting key stemness markers, as confirmed by co-localization studies. Moreover, the drug combination significantly decreases the growth of various PC patient-derived organoids (P < 0.001). We found that SOX9 is significantly overexpressed in high-grade PC tumors (P < 0.05) and in chemotherapy-treated patients compared to chemo-naïve patients (P < 0.05). These results were further validated using publicly available datasets. Moreover, afatinib alone or in combination with gemcitabine decreased stemness and tumorspheres by reducing phosphorylation of EGFR family proteins, ERK, FAK, and CSC markers. Mechanistically, afatinib treatment decreased CSC markers by downregulating SOX9 via FOXA2. Indeed, EGFR and FOXA2 depletion reduced SOX9 expression in PCSCs. Taken together, pan-EGFR inhibition by afatinib impedes PCSCs growth and metastasis via the EGFR/ERK/FOXA2/SOX9 axis. This novel mechanism of pan-EGFR inhibitor and its ability to eradicate CSC may serve as a tailor-made approach to enhance chemotherapeutic benefits in other cancer types.
Asunto(s)
Factor Nuclear 3-beta del Hepatocito/antagonistas & inhibidores , Neoplasias Pancreáticas/tratamiento farmacológico , Factor de Transcripción SOX9/antagonistas & inhibidores , Afatinib/uso terapéutico , Animales , Línea Celular Tumoral , Desoxicitidina/análogos & derivados , Desoxicitidina/uso terapéutico , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/fisiología , Factor Nuclear 3-beta del Hepatocito/fisiología , Humanos , Ratones , Metástasis de la Neoplasia , Células Madre Neoplásicas/efectos de los fármacos , Neoplasias Pancreáticas/patología , Factor de Transcripción SOX9/fisiología , GemcitabinaRESUMEN
Studies in vertebrates have outlined conserved molecular control of definitive endoderm (END) development. However, recent work also shows that key molecular aspects of human END regulation differ even from rodents. Differentiation of human embryonic stem cells (ESCs) to END offers a tractable system to study the molecular basis of normal and defective human-specific END development. Here, we interrogated dynamics in chromatin accessibility during differentiation of ESCs to END, predicting DNA-binding proteins that may drive this cell fate transition. We then combined single-cell RNA-seq with parallel CRISPR perturbations to comprehensively define the loss-of-function phenotype of those factors in END development. Following a few candidates, we revealed distinct impairments in the differentiation trajectories for mediators of TGFß signaling and expose a role for the FOXA2 transcription factor in priming human END competence for human foregut and hepatic END specification. Together, this single-cell functional genomics study provides high-resolution insight on human END development.
Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , ARN Guía de Kinetoplastida/metabolismo , Factores de Transcripción/metabolismo , Diferenciación Celular , Cromatina/metabolismo , Endodermo/citología , Endodermo/metabolismo , Factor Nuclear 3-beta del Hepatocito/antagonistas & inhibidores , Factor Nuclear 3-beta del Hepatocito/genética , Factor Nuclear 3-beta del Hepatocito/metabolismo , Células Madre Embrionarias Humanas/citología , Células Madre Embrionarias Humanas/metabolismo , Humanos , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Interferencia de ARN , Factores de Transcripción SOXF/genética , Factores de Transcripción SOXF/metabolismo , Transducción de Señal , Análisis de la Célula Individual , Proteína Smad4/genética , Proteína Smad4/metabolismo , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genética , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Forkhead box protein A2 (FOXA2) is a core transcription factor that controls cell differentiation and may have an important role in bone metabolism. However, the role of FOXA2 during osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) remains largely unknown. In this study, decreased expression of FOXA2 was observed during osteogenic differentiation of rat BMSCs (rBMSCs). FOXA2 knockdown significantly increased osteoblast-specific gene expression, the number of mineral deposits and alkaline phosphatase activity, whereas FOXA2 overexpression inhibited osteogenesis-specific activities. Moreover, extracellular signal-regulated protein kinase (ERK) signalling was upregulated following knockdown of FOXA2. The enhanced osteogenesis due to FOXA2 knockdown was partially rescued by an ERK inhibitor. Using a rat tibial defect model, a rBMSC sheet containing knocked down FOXA2 significantly improved bone healing. Collectively, these findings indicated that FOXA2 had an essential role in osteogenic differentiation of BMSCs, partly by activation of the ERK signalling pathway.
Asunto(s)
Diferenciación Celular , Factor Nuclear 3-beta del Hepatocito/metabolismo , Sistema de Señalización de MAP Quinasas , Animales , Células de la Médula Ósea/citología , Butadienos/farmacología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Fracturas Óseas/patología , Fracturas Óseas/terapia , Fracturas Óseas/veterinaria , Factor Nuclear 3-beta del Hepatocito/antagonistas & inhibidores , Factor Nuclear 3-beta del Hepatocito/genética , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Nitrilos/farmacología , Osteoblastos/citología , Osteoblastos/metabolismo , Osteogénesis , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Ratas , Ratas Sprague-Dawley , Andamios del Tejido/químicaRESUMEN
MicroRNAs (miRNAs) play an active role in the pathogenesis of gastric cancer. The expression and biological function for miR-187 in gastric cancer remains unknow. In the present study, we demonstrated that miR-187 expression was increased in gastric cancer (GC) tissues and cells. Increased expression level of miR-187 was associated with adverse clinical features including tumor size, lymph metastasis and TNM stage, and decreased overall survival and disease-free survival of GC patients. Functionally, overexpression miR-187 could promote while inhibition of miR-187 could suppress, the proliferation, migration and invasion of GC cells in vitro. In vivo experiments showed that overexpression of miR-187 promoted the growth and lung metastasis of SGC-7901 cells in nude mice. Mechanically, we confirmed that FOXA2 was the downstream target of miR-187 in GC cells using luciferase assay, qRT-PCR and western blot analysis. Moreover, overexpression of FOXA2 abrogated the promoting effects of miR-187 overexpression on SGC-7901 cell proliferation, migration and invasion, while inhibition of FOXA2 reversed the inhibitory effects of miR-187 downregulation on these biological functions of AGS cells, suggesting that FOXA2 was a functional mediator of miR-187 in GC. Therefore, this study indicates that miR-187 is potentially a biomarker and treatment target for GC patients.
Asunto(s)
Movimiento Celular , Proliferación Celular , Factor Nuclear 3-beta del Hepatocito/metabolismo , MicroARNs/genética , Neoplasias Gástricas/patología , Anciano , Animales , Apoptosis , Western Blotting , Línea Celular Tumoral , Femenino , Mucosa Gástrica/metabolismo , Regulación Neoplásica de la Expresión Génica , Factor Nuclear 3-beta del Hepatocito/antagonistas & inhibidores , Factor Nuclear 3-beta del Hepatocito/genética , Humanos , Metástasis Linfática , Masculino , Ratones , Ratones Desnudos , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Estómago/patología , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The mechanisms that allow breast cancer (BCa) cells to metabolically sustain rapid growth are poorly understood. Here we report that BCa cells are dependent on a mechanism to supply precursors for intracellular lipid production derived from extracellular sources and that the endothelial lipase (LIPG) fulfils this function. LIPG expression allows the import of lipid precursors, thereby contributing to BCa proliferation. LIPG stands out as an essential component of the lipid metabolic adaptations that BCa cells, and not normal tissue, must undergo to support high proliferation rates. LIPG is ubiquitously and highly expressed under the control of FoxA1 or FoxA2 in all BCa subtypes. The downregulation of either LIPG or FoxA in transformed cells results in decreased proliferation and impaired synthesis of intracellular lipids.
Asunto(s)
Neoplasias de la Mama/metabolismo , Regulación Neoplásica de la Expresión Génica , Factor Nuclear 3-alfa del Hepatocito/metabolismo , Factor Nuclear 3-beta del Hepatocito/metabolismo , Lipasa/metabolismo , Metabolismo de los Lípidos/genética , 4-Butirolactona/análogos & derivados , 4-Butirolactona/farmacología , Animales , Transporte Biológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Doxiciclina/farmacología , Inhibidores Enzimáticos/farmacología , Femenino , Factor Nuclear 3-alfa del Hepatocito/antagonistas & inhibidores , Factor Nuclear 3-alfa del Hepatocito/genética , Factor Nuclear 3-beta del Hepatocito/antagonistas & inhibidores , Factor Nuclear 3-beta del Hepatocito/genética , Humanos , Lactonas/farmacología , Lipasa/antagonistas & inhibidores , Lipasa/genética , Células MCF-7 , Ratones , Ratones Desnudos , Invasividad Neoplásica , Orlistat , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The Forkhead box A transcription factors are major regulators of glucose homeostasis. They show both distinct and redundant roles during pancreas development and in adult mouse ß-cells. In vivo ablation studies have revealed critical implications of Foxa1 on glucagon biosynthesis and requirement of Foxa2 in α-cell terminal differentiation. In order to examine the respective role of these factors in mature α-cells, we used small interfering RNA (siRNA) directed against Foxa1 and Foxa2 in rat primary pancreatic α-cells and rodent α-cell lines leading to marked decreases in Foxa1 and Foxa2 mRNA levels and proteins. Both Foxa1 and Foxa2 control glucagon gene expression specifically through the G2 element. Although we found that Foxa2 controls the expression of the glucagon, MafB, Pou3f4, Pcsk2, Nkx2.2, Kir6.2, and Sur1 genes, Foxa1 only regulates glucagon gene expression. Interestingly, the Isl1 and Gipr genes were not controlled by either Foxa1 or Foxa2 alone but by their combination. Foxa1 and Foxa2 directly activate and bind the promoter region the Nkx2.2, Kir6.2 and Sur1, Gipr, Isl1, and Pou3f4 genes. We also demonstrated that glucagon secretion is affected by the combined effects of Foxa1 and Foxa2 but not by either one alone. Our results indicate that Foxa1 and Foxa2 control glucagon biosynthesis and secretion as well as α-cell differentiation with both common and unique target genes.
Asunto(s)
Diferenciación Celular/genética , Células Secretoras de Glucagón/fisiología , Glucagón/biosíntesis , Glucagón/metabolismo , Factor Nuclear 3-alfa del Hepatocito/fisiología , Factor Nuclear 3-beta del Hepatocito/fisiología , Animales , Sitios de Unión/genética , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Cricetinae , Factor Nuclear 3-alfa del Hepatocito/antagonistas & inhibidores , Factor Nuclear 3-beta del Hepatocito/antagonistas & inhibidores , Proteína Homeobox Nkx-2.2 , Masculino , Regiones Promotoras Genéticas , ARN Interferente Pequeño/farmacología , RatasRESUMEN
UNLABELLED: Hepatocyte apoptosis is a ubiquitous feature of chronic liver injury, but the molecular mechanism remains to be determined. The liver-enriched Foxa2 transcription factor has been implicated in inflammation and neoplasia. Foxa2 may play a role in the regulation of apoptosis. This study aimed to investigate the relationship between Foxa2 and hepatic apoptosis. Apoptosis was induced with different causative factors as measured by caspase activity and TUNEL assay. Results showed that the apoptotic injury was associated with a downregulation of Foxa2. Foxa2-expressing vectors decreased apoptosis, whereas siRNA silencing of Foxa2 increased apoptosis in HepG2 cells. Foxa2 was correlated with expression profiling of anti-apoptotic genes cIAP1, cIAP2, XIAP, and survivin. Significantly, the cIAP1 expression was decreased by siRNA silencing of Foxa2, but increased by Foxa2-expressing vectors. The promoter of cIAP1 had specific DNA sequences that could be bound by Foxa2 nuclear protein as demonstrated by EMSA and gel supershift assay. The cIAP1 promoter was also occupied by Foxa2 nuclear factor through ChIP assay. Deletion of putative Foxa2 binding domains in cIAP1 promoter significantly reduced its promoter activity. CONCLUSION: A mechanism by which Foxa2 transcription factor modulates hepatic apoptosis may be through cIAP1 signaling pathway. Foxa2 can be a potential target for therapeutic intervention in liver diseases.
Asunto(s)
Apoptosis , Factor Nuclear 3-beta del Hepatocito/metabolismo , Proteínas Inhibidoras de la Apoptosis/metabolismo , Animales , Células Cultivadas , Regulación hacia Abajo , Ensayo de Cambio de Movilidad Electroforética , Células Hep G2 , Factor Nuclear 3-beta del Hepatocito/antagonistas & inhibidores , Factor Nuclear 3-beta del Hepatocito/genética , Hepatocitos/citología , Hepatocitos/metabolismo , Humanos , Proteínas Inhibidoras de la Apoptosis/genética , Hígado/lesiones , Ratones , Ratones Endogámicos C57BL , Regiones Promotoras Genéticas , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Survivin , Proteína Inhibidora de la Apoptosis Ligada a X/genética , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismoRESUMEN
OBJECTIVE: We aimed to investigate whether vascular endothelial growth factor (VEGF) influences apolipoprotein M (ApoM) expression and pre-ß-high-density lipoprotin (HDL) formation, and whether forkhead box A2 (Foxa2) and Nur77 are involved in this process. METHODS AND RESULTS: We analyzed the serum VEGF concentrations of 264 adults who underwent a medical checkup and found that VEGF concentration was positively correlated with serum triglyceride, total cholesterol, LDL cholesterol (LDL-C), very-low-density lipoprotein cholesterol (VLDL-C), and ApoB concentrations, but was negatively correlated with serum high-density lipoprotein cholesterol (HDL-C) and ApoM concentrations. We further investigated the effects of VEGF on ApoM expression and pre-ß-HDL formation, and the mechanisms responsible, in HepG2 cells and mouse primary hepatocytes. VEGF markedly downregulated ApoM expression and pre-ß-HDL formation. At the same time, expression of Foxa2 was also inhibited, whereas expression of Nur77 was increased by treatment with VEGF. Furthermore, small interfering (si) RNA knockdown of Foxa2 made the downregulation of VEGF on ApoM expression and pre-ß-HDL formation even more obvious. In addition, siRNA knockdown of Nur77 significantly compensated for the inhibitory effect of VEGF on Foxa2 expression, whereas the Nur77 agonist cytosporone B led to the downregulation of Foxa2 expression more significantly than VEGF. Moreover, overexpression of a Nur77 transgene in C57BL/6 mice resulted in decreased serum ApoM and pre-ß-HDL levels, whereas si-Nur77-treated mice displayed upregulated serum ApoM and pre-ß-HDL levels. CONCLUSION: These results provide evidence that VEGF may first downregulate expression of Foxa2 by enhancing Nur77 activity and then decrease expression of ApoM and pre-ß-HDL formation. Therefore, our study may be useful in understanding the critical effect of VEGF in the pathogenesis of atherosclerosis.
Asunto(s)
Apolipoproteínas/genética , Regulación hacia Abajo , Factor Nuclear 3-beta del Hepatocito/antagonistas & inhibidores , Lipocalinas/genética , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Adolescente , Adulto , Anciano , Animales , Apolipoproteínas/metabolismo , Apolipoproteínas M , Niño , Femenino , Factor Nuclear 3-beta del Hepatocito/genética , Factor Nuclear 3-beta del Hepatocito/metabolismo , Hepatocitos/metabolismo , Humanos , Lipocalinas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/antagonistas & inhibidores , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/metabolismo , ARN Interferente Pequeño/metabolismoRESUMEN
Type 1 iodothyronine deiodinase (D1), a selenoenzyme that catalyzes the bioactivation of thyroid hormone, is expressed mainly in the liver. Its expression and activity are modulated by several factors, but the precise mechanism of its transcriptional regulation remains unclear. In the present study, we have analyzed the promoter of human D1 gene (hDIO1) to identify factors that prevalently increase D1 activity in the human liver. Deletion and mutation analyses demonstrated that a forkhead box (FOX)A binding site and an E-box site within the region between nucleotides -187 and -132 are important for hDIO1 promoter activity in the liver. EMSA demonstrated that FOXA1 and FOXA2 specifically bind to the FOXA binding site and that upstream stimulatory factor (USF) specifically binds to the E-box element. Overexpression of FOXA2 decreased hDIO1 promoter activity, and short interfering RNA-mediated knockdown of FOXA2 increased the expression of hDIO1 mRNA. In contrast, overexpression of USF1/2 increased hDIO1 promoter activity. Short interfering RNA-mediated knockdown of FOXA1 decreased the expression of hDIO1 mRNA, but knockdown of both FOXA1 and FOXA2 restored it. The response of the hDIO1 promoter to USF was greatly attenuated in the absence of FOXA1. Taken together, these results indicate that a balance of FOXA1 and FOXA2 expression modulates hDIO1 expression in the liver.
Asunto(s)
Proteínas de Unión al ADN/genética , Factor Nuclear 3-alfa del Hepatocito/metabolismo , Factor Nuclear 3-beta del Hepatocito/metabolismo , Hígado/metabolismo , Región de Flanqueo 5' , Secuencia de Bases , Sitios de Unión/genética , Cartilla de ADN/genética , Elementos E-Box , Regulación Enzimológica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Células Hep G2 , Factor Nuclear 3-alfa del Hepatocito/antagonistas & inhibidores , Factor Nuclear 3-alfa del Hepatocito/genética , Factor Nuclear 3-beta del Hepatocito/antagonistas & inhibidores , Factor Nuclear 3-beta del Hepatocito/genética , Humanos , Modelos Biológicos , Regiones Promotoras Genéticas , ARN Interferente Pequeño/genética , Factores Estimuladores hacia 5'/genética , Factores Estimuladores hacia 5'/metabolismoRESUMEN
Recently, we demonstrated that the transcription factors HNF6 and FOXA2 function as key regulators in human colorectal liver metastases. To better understand their proposed inhibitory crosstalk, the consequences of functional knockdown of FOXA2 on HNF6 and C/EBPα activity were investigated in the human colon Caco-2 and HepG2 carcinoma cell lines. Specifically, siRNA-mediated gene silencing of FOXA2 repressed transcript expression by >80%. This resulted in a statistically significant 6-, 3-, 4-, and 8-fold increase in mRNA expression of HNF6 and of genes targeted by this transcription factor, e.g., HSP105B, CYP51, and C/EBPα, as determined by qRT-PCR. Thus, functional knockdown of FOXA2 recovered HNF6 activity. Furthermore, with nuclear extracts of Caco-2 cells no HNF6 DNA binding was observed, but expression of HNF1α, FOXA2, FOXA3, and HNF4α protein was abundant. We therefore transfected a plasmid encoding HNF6 into Caco-2 cells but also employed a retroviral vector to transfect HNF6 into HepG2 cells. This resulted in HNF6 protein expression with DNA binding activity being recovered as determined by EMSA band shift assays. Furthermore, by flow cytometry the consequences of HNF6 expression on cell cycle regulation in transfected cells was studied. Essentially, HNF6 inhibited cell cycle progression in the G2/M and G1 phase in Caco-2 and HepG2 cell lines, respectively. Here, proliferation was reduced by 80% and 50% in Caco-2 and HepG2 cells, respectively, as determined by the BrdU labeling assay. Therefore functional knockdown of FOXA2 recovered HNF6 activity and inhibited growth of tumor-cells and may possibly represent a novel therapeutic target in primary and secondary liver malignancies.
Asunto(s)
Carcinoma Hepatocelular/fisiopatología , Neoplasias del Colon/fisiopatología , Factor Nuclear 3-beta del Hepatocito/antagonistas & inhibidores , Factor Nuclear 6 del Hepatocito/fisiología , Neoplasias Hepáticas/fisiopatología , Western Blotting , Carcinoma Hepatocelular/patología , Ciclo Celular , Línea Celular Tumoral , Proliferación Celular , Neoplasias del Colon/patología , Ensayo de Cambio de Movilidad Electroforética , Técnicas de Silenciamiento del Gen , Vectores Genéticos , Factor Nuclear 3-beta del Hepatocito/genética , Factor Nuclear 6 del Hepatocito/genética , Humanos , Neoplasias Hepáticas/patología , Interferencia de ARN , ARN Interferente Pequeño , Retroviridae/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TransfecciónRESUMEN
The expression of Sox14 gene in spinal cord explants was found to be regulated by Sonic hedgehog (SHH) in a dose-dependent manner, indicating that this signaling molecule might act as a regulator of Sox14-expressing interneuron differentiation. In the present study we identified the positive control element and provided the first evidence that FOXA2 is involved in up-regulation of SOX14 expression in HepG2 and U87MG cell lines. By functional analysis we demonstrated that mutation in FOXA2 binding site reduced the SOX14 reporter construct activity, and that FOXA2 over-expression increased endogenous SOX14 protein expression. Further, we have shown that human SOX14 expression is GLI1 dependent in U87MG cells and SHH-N dependent in U87MG and HepG2 cell lines. By applying siRNA silencing of FOXA2, we have demonstrated that upregulation of endogenous SOX14 gene expression by SHH is, at least in part, mediated by FOXA2. However, our data revealed that a positive regulatory region, containing functional FOXA2 site analyzed in this study, is not involved in mediation of SHH dependent SOX14 activation. Data presented here provide the initial insight into molecular mechanism underlying tissue and developmentally specific regulation of the SOX14 gene expression.
Asunto(s)
Factor Nuclear 3-beta del Hepatocito/metabolismo , Factores de Transcripción SOXB2/genética , Animales , Secuencia de Bases , Sitios de Unión/genética , Línea Celular , Cartilla de ADN/genética , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Factor Nuclear 3-beta del Hepatocito/antagonistas & inhibidores , Factor Nuclear 3-beta del Hepatocito/genética , Humanos , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Ratones , Mutagénesis Sitio-Dirigida , ARN Interferente Pequeño/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos , Distribución Tisular , Transfección , Regulación hacia Arriba , Proteína con Dedos de Zinc GLI1RESUMEN
Foxa2 (HNF3 beta) is a one of three, closely related transcription factors that are critical to the development and function of the mouse liver. We have used chromatin immunoprecipitation and massively parallel Illumina 1G sequencing (ChIP-Seq) to create a genome-wide profile of in vivo Foxa2-binding sites in the adult liver. More than 65% of the approximately 11.5 k genomic sites associated with Foxa2 binding, mapped to extended gene regions of annotated genes, while more than 30% of intragenic sites were located within first introns. 20.5% of all sites were further than 50 kb from any annotated gene, suggesting an association with novel gene regions. QPCR analysis demonstrated a strong positive correlation between peak height and fold enrichment for Foxa2-binding sites. We measured the relationship between Foxa2 and liver gene expression by overlapping Foxa2-binding sites with a SAGE transcriptome profile, and found that 43.5% of genes expressed in the liver were also associated with Foxa2 binding. We also identified potential Foxa2-interacting transcription factors whose motifs were enriched near Foxa2-binding sites. Our comprehensive results for in vivo Foxa2-binding sites in the mouse liver will contribute to resolving transcriptional regulatory networks that are important for adult liver function.
Asunto(s)
Factor Nuclear 3-beta del Hepatocito/metabolismo , Hígado/metabolismo , Elementos Reguladores de la Transcripción , Animales , Sitios de Unión , Inmunoprecipitación de Cromatina , Biología Computacional , Femenino , Expresión Génica , Redes Reguladoras de Genes , Genómica , Factor Nuclear 3-beta del Hepatocito/antagonistas & inhibidores , Ratones , Ratones Endogámicos C57BL , Interferencia de ARN , Análisis de Secuencia de ADN , Factores de Transcripción/metabolismoRESUMEN
The mucin Muc2 is the main component of the intestinal mucus layer and thus plays important roles in intestinal protection. Therefore, it is important to understand its regulation during goblet cell differentiation. Foxa1 and Foxa2 forkhead box transcription factors (TFs) participate in transcriptional programs governing intestinal cell differentiation. Using immunohistochemistry, we showed a spatio-temporal pattern of expression of both TFs in developing and adult mouse intestine and their expression in Muc2-expressing intestinal cells. Down-regulation of Foxa1 and Foxa2 by RNA interference in cultured intestinal cells decreased Muc2 mRNA level by half, and abolished Muc2 protein expression. Chromatin immunoprecipitation and gel shift assays showed that these two TFs directly bind to the Muc2 promoter. Co-transfection experiments indicated that both TFs activate the Muc2 promoter and that mutations of three Foxa cis-elements inhibit Muc2 transactivation. In conclusion, this work identifies Foxa1 and Foxa2 as important regulators of Muc2 expression in the intestine.
Asunto(s)
Células Caliciformes/metabolismo , Factor Nuclear 3-alfa del Hepatocito/metabolismo , Factor Nuclear 3-beta del Hepatocito/metabolismo , Intestino Delgado/metabolismo , Mucinas/genética , Activación Transcripcional , Animales , Diferenciación Celular , Inmunoprecipitación de Cromatina , Ensayo de Cambio de Movilidad Electroforética , Factor de Transcripción GATA4/metabolismo , Células Caliciformes/citología , Factor Nuclear 3-alfa del Hepatocito/antagonistas & inhibidores , Factor Nuclear 3-beta del Hepatocito/antagonistas & inhibidores , Intestino Delgado/citología , Ratones , Ratones Endogámicos BALB C , Mucina 2 , Mucinas/metabolismo , Mutación , Regiones Promotoras Genéticas , Interferencia de ARN , Activación Transcripcional/genéticaRESUMEN
Low levels of protein C (PC) predict outcome as early as 10 h after insult in a rat polymicrobial sepsis model and were associated with suppression of PC mRNA, upstream transcription factor FoxA2, and cofactor hepatocyte nuclear factor 6 (HNF6). Small interfering RNA suppression of FoxA2 in isolated hepatocytes demonstrated regulation of both its cofactor HNF6 and PC. Our data suggest that reduced FoxA2 may be important in the suppression of PC and resulting poor outcome in sepsis.
Asunto(s)
Factor Nuclear 3-beta del Hepatocito/fisiología , Proteína C/antagonistas & inhibidores , Proteína C/biosíntesis , Sepsis/metabolismo , Enfermedad Aguda , Animales , Secuencia de Bases , Biomarcadores , Ciego , Modelos Animales de Enfermedad , Femenino , Factor Nuclear 3-beta del Hepatocito/antagonistas & inhibidores , Factor Nuclear 3-beta del Hepatocito/genética , Ligadura , Datos de Secuencia Molecular , Valor Predictivo de las Pruebas , Pronóstico , Proteína C/genética , Proteína C/fisiología , Punciones , ARN Mensajero/antagonistas & inhibidores , ARN Mensajero/biosíntesis , ARN Interferente Pequeño/farmacología , Ratas , Ratas Sprague-Dawley , Sepsis/diagnóstico , Sepsis/mortalidadRESUMEN
VLDL levels are elevated in type II diabetes, where they contribute to the risk of coronary heart disease. A study by Wolfrum and Stoffel (2006) shows that the forkhead protein Foxa2 stimulates hepatic VLDL production in concert with the coactivator PGC-1beta and that insulin inhibits this process by inactivating Foxa2.