RESUMEN
HNF4α is a nuclear receptor produced as 12 isoforms from two promoters by alternative splicing. To characterize the transcriptional capacities of all 12 HNF4α isoforms, stable lines expressing each isoform were generated. The entire transcriptome associated with each isoform was analyzed as well as their respective interacting proteome. Major differences were noted in the transcriptional function of these isoforms. The α1 and α2 isoforms were the strongest regulators of gene expression whereas the α3 isoform exhibited significantly reduced activity. The α4, α5, and α6 isoforms, which use an alternative first exon, were characterized for the first time, and showed a greatly reduced transcriptional potential with an inability to recognize the consensus response element of HNF4α. Several transcription factors and coregulators were identified as potential specific partners for certain HNF4α isoforms. An analysis integrating the vast amount of omics data enabled the identification of transcriptional regulatory mechanisms specific to certain HNF4α isoforms, hence demonstrating the importance of considering all isoforms given their seemingly diverse functions.
Asunto(s)
Factor Nuclear 4 del Hepatocito/metabolismo , Transcripción Genética , Línea Celular Tumoral , ADN/metabolismo , Regulación de la Expresión Génica , Redes Reguladoras de Genes , Proteínas Fluorescentes Verdes/metabolismo , Factor Nuclear 4 del Hepatocito/genética , Humanos , Unión Proteica , Isoformas de Proteínas/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Elementos de Respuesta/genética , Factores de Transcripción/metabolismo , Transcriptoma/genéticaRESUMEN
HNF4α is a key nuclear receptor for regulating gene expression in the gut. Although both P1 and P2 isoform classes of HNF4α are expressed in colonic epithelium, specific inhibition of P1 isoforms is commonly found in colorectal cancer. Previous studies have suggested that P1 and P2 isoforms might regulate different cellular functions. Despite these advances, it remains unclear whether these isoform classes are functionally divergent in the context of human biology. Here, the consequences of specific inhibition of P1 or P2 isoform expression was measured in a human colorectal cancer cell transcriptome. Results indicate that P1 isoforms were specifically associated with the control of cell metabolism, whereas P2 isoforms globally supported aberrant oncogenic signalization, promoting cancer cell survival and progression. P1 promoter-driven isoform expression was found to be repressed by ß-catenin, one of the earliest oncogenic pathways to be activated during colon tumorigenesis. These findings identify a novel cascade by which the expression of P1 isoforms is rapidly shut down in the early stages of colon tumorigenesis, allowing a change in HNF4α-dependent transcriptome, thereby promoting colorectal cancer progression.This article has an associated First Person interview with the first author of the paper.
Asunto(s)
Neoplasias Colorrectales/metabolismo , Factor Nuclear 4 del Hepatocito/genética , Regiones Promotoras Genéticas , beta Catenina/genética , Animales , Línea Celular Tumoral , Neoplasias Colorrectales/genética , Regulación Neoplásica de la Expresión Génica , Factor Nuclear 4 del Hepatocito/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transcriptoma , beta Catenina/metabolismoRESUMEN
Colorectal tumors are immersed in an array of tumor-promoting factors including extracellular nucleotides such as uridine 5'diphosphate (UDP). UDP is the endogenous agonist of the G protein-coupled P2Y6 receptor (P2Y6R), which may contribute to the formation of a tumor-promoting microenvironment by coordinating resistance to apoptosis. Colorectal cancer (CRC) was chemically induced in P2ry6 knockout (P2ry6-/-) mice using azoxymethane and dextran sulfate sodium challenges. Mice were euthanatized and their tumor load determined. Fixed tissues were stained for histological and immunohistochemistry analysis. Tumoroids were also prepared from CRC tumors resected from P2ry6+/+ mice to determine the role of P2Y6R in resistance to apoptosis, whereas HT29 carcinoma cells were used to elucidate the signaling mechanism involved in P2Y6R anti-apoptotic effect. P2ry6-/- mice developed a reduced number of colorectal tumors with apparent tumors having smaller volumes. Overall dysplastic score was significantly lower in P2ry6-/- animals. Stimulation of P2Y6R with the selective agonist MRS2693 protected HT-29 cells from TNFα-induced apoptosis. This protective effect was mediated by the stabilizing phosphorylation of the X-linked inhibitor of apoptosis protein (XIAP) by AKT. Using CRC-derived tumoroids, P2Y6R activation was found to contribute to chemoresistance since addition of the P2Y6R agonist MRS2693 significantly prevented the cytotoxic effect of 5-fluorouracil. The present study shows that sustained activation of P2Y6R may contribute to intestinal tumorigenesis by blocking the apoptotic process and by contributing to chemoresistance, a substantial concern in the treatment of patients with CRC. These results suggest that P2Y6R may represent a prime target for reducing colorectal carcinogenesis.
Asunto(s)
Apoptosis , Transformación Celular Neoplásica/metabolismo , Neoplasias Colorrectales/metabolismo , Proteínas de Neoplasias/metabolismo , Receptores Purinérgicos P2/metabolismo , Animales , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Femenino , Humanos , Masculino , Ratones , Ratones Noqueados , Proteínas de Neoplasias/genética , Receptores Purinérgicos P2/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The elevated expression of the splicing regulator SRSF10 in metastatic colorectal cancer (CRC) stimulates the production of the pro-tumorigenic BCLAF1-L splice variant. We discovered a group of small molecules with an aminothiazole carboxamide core (GPS167, GPS192 and others) that decrease production of BCLAF1-L. While additional alternative splicing events regulated by SRSF10 are affected by GPS167/192 in HCT116 cells (e.g. in MDM4, WTAP, SLK1 and CLK1), other events are shifted in a SRSF10-independent manner (e.g. in MDM2, NAB2 and TRA2A). GPS167/192 increased the interaction of SRSF10 with the CLK1 and CLK4 kinases, leading us to show that GPS167/192 can inhibit CLK kinases preferentially impacting the activity of SRSF10. Notably, GPS167 impairs the growth of CRC cell lines and organoids, inhibits anchorage-independent colony formation, cell migration, and promotes cytoxicity in a manner that requires SRSF10 and p53. In contrast, GPS167 only minimally affects normal colonocytes and normal colorectal organoids. Thus, GPS167 reprograms the tumorigenic activity of SRSF10 in CRC cells to elicit p53-dependent apoptosis.
RESUMEN
Hepatocyte nuclear factor 4alpha (HNF4alpha) is a regulator of hepatocyte and pancreatic transcription. Hnf4alpha deletion in the mouse is embryonically lethal with severe defects in visceral endoderm formation. It has been concluded in the past that the role of Hnf4alpha in the developing colon was much less important than in the liver. However, the precise role of Hnf4alpha in the homeostasis of the small intestinal epithelium remains unclear. Our aim was to evaluate the potential of Hnf4alpha to support an intestinal epithelial phenotype. First, Hnf4alpha potential to dictate this phenotype was assessed in nonintestinal cell lines in vitro. Forced expression of Hnf4alpha in fibroblasts showed an induction of features normally restricted to epithelial cells. Combinatory expression of Hnf4alpha with specific transcriptional regulators of the intestine resulted in the induction of intestinal epithelial genes in this context. Second, the importance of Hnf4alpha in maintaining the homeostasis of the intestinal epithelium was investigated in mice. Mice conditionally deficient for intestinal Hnf4alpha developed normally throughout adulthood with an epithelium displaying normal morphological and functional structures with minor alterations. Subtle but statistical differences were observed at the proliferation and the cytodifferentiation levels. Hnf4alpha mutant mice displayed an increase in the number of goblet and enteroendocrine cells compared with controls. Given the fundamental role of this transcription factor in other tissues, these findings dispute the crucial role for this regulator in the maintenance of intestinal epithelial cell function at a period of time that follows cytodifferentiation but may suggest a functional role in instructing cells to become specific to the intestinal epithelium.
Asunto(s)
Diferenciación Celular , Células Epiteliales/metabolismo , Factor Nuclear 4 del Hepatocito/metabolismo , Mucosa Intestinal/metabolismo , Envejecimiento/metabolismo , Animales , Factor de Transcripción CDX2 , Células CACO-2 , Proliferación Celular , Forma de la Célula , Células Epiteliales/diagnóstico por imagen , Factor de Transcripción GATA4/genética , Factor de Transcripción GATA4/metabolismo , Factor Nuclear 4 del Hepatocito/deficiencia , Factor Nuclear 4 del Hepatocito/genética , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Homeostasis , Humanos , Integrasas/genética , Mucosa Intestinal/ultraestructura , Ratones , Ratones Noqueados , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Células 3T3 NIH , Fenotipo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transfección , UltrasonografíaRESUMEN
Hepatocyte nuclear factor 4α (HNF4α) is a transcription factor that acts as a master regulator of genes for several endoderm-derived tissues, including the intestine, in which it plays a central role during development and tumorigenesis. To better define the mechanisms by which HNF4α can influence these processes, we identified proteins interacting with HNF4α using stable isotope labelling with amino acids in cell culture (SILAC)-based quantitative proteomics with either immunoprecipitation of green fluorescent protein (GFP) or with proximity-dependent purification by the biotin ligase BirA (BioID), both fused to HNF4α. Surprisingly, these analyses identified a significant enrichment of proteins characterized with a role in DNA repair, a so far unidentified biological feature of this transcription factor. Several of these proteins including PARP1, RAD50, and DNA-PKcs were confirmed to interact with HNF4α in colorectal cancer cell lines. Following DNA damage, HNF4α was able to increase cell viability in colorectal cancer cells. Overall, these observations identify a potential role for this transcription factor during the DNA damage response.
RESUMEN
Bmps are morphogens involved in various gastric cellular functions. Studies in genetically-modified mice have shown that Bmp disruption in gastric epithelial and stromal cell compartments leads to the development of tumorigenesis. Our studies have demonstrated that abrogation of gastric epithelial Bmp signaling alone was not sufficient to recapitulate the neoplastic features associated with total gastric loss of Bmp signaling. Thus, epithelial Bmp signaling does not appear to be a key player in gastric tumorigenesis initiation. These observations suggest a greater role for stromal Bmp signaling in gastric polyposis initiation. In order to identify the specific roles played by mesenchymal Bmp signaling in gastric homeostasis, we generated a mouse model with abrogation of Bmp signaling exclusively in the gastro-intestinal mesenchyme (Bmpr1a(ΔMES)). We were able to expose an unsuspected role for Bmp loss of signaling in leading normal gastric mesenchyme to adapt into reactive mesenchyme. An increase in the population of activated-fibroblasts, suggesting mesenchymal transdifferentiation, was observed in mutant stomach. Bmpr1a(ΔMES) stomachs exhibited spontaneous benign polyps with presence of both intestinal metaplasia and spasmolytic-polypeptide-expressing metaplasia as early as 90 days postnatal. These results support the novel concept that loss of mesenchymal Bmp signaling cascade acts as a trigger in gastric polyposis initiation.
Asunto(s)
Pólipos Adenomatosos/genética , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/genética , Proteínas Morfogenéticas Óseas/metabolismo , Transformación Celular Neoplásica/genética , Neoplasias Gástricas/genética , Células del Estroma/metabolismo , Pólipos Adenomatosos/metabolismo , Animales , Diferenciación Celular , Proliferación Celular , Transformación Celular Neoplásica/metabolismo , Regulación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Ratones , Transducción de Señal , Neoplasias Gástricas/metabolismoRESUMEN
Hepatocyte nuclear factor 4-alpha (HNF4-α) is a nuclear receptor regulating metabolism, cell junctions, differentiation and proliferation in liver and intestinal epithelial cells. Mutations within the HNF4A gene are associated with human diseases such as maturity-onset diabetes of the young. Recently, HNF4A has also been described as a susceptibility gene for ulcerative colitis in genome-wide association studies. In addition, specific HNF4A genetic variants have been identified in pediatric cohorts of Crohn's disease. Results obtained from knockout mice supported that HNF4-α can protect the intestinal mucosae against inflammation. However, the exact molecular links behind HNF4-α and inflammatory bowel diseases remains elusive. In this review, we will summarize the current knowledge about the role of HNF4-α and its isoforms in inflammation. Specific nature of HNF4-α P1 and P2 classes of isoforms will be summarized. HNF4-α role as a hepatocyte mediator for cytokines relays during liver inflammation will be integrated based on documented examples of the literature. Conclusions that can be made from these earlier liver studies will serve as a basis to extrapolate correlations and divergences applicable to intestinal inflammation. Finally, potential functional roles for HNF4-α isoforms in protecting the intestinal mucosae from chronic and pathological inflammation will be presented.
Asunto(s)
Hepatitis/metabolismo , Factor Nuclear 4 del Hepatocito/metabolismo , Hepatocitos/metabolismo , Mediadores de Inflamación/metabolismo , Enfermedades Inflamatorias del Intestino/metabolismo , Mucosa Intestinal/metabolismo , Hígado/metabolismo , Transducción de Señal , Animales , Hepatitis/genética , Hepatitis/inmunología , Factor Nuclear 4 del Hepatocito/genética , Hepatocitos/inmunología , Humanos , Enfermedades Inflamatorias del Intestino/genética , Enfermedades Inflamatorias del Intestino/inmunología , Mucosa Intestinal/inmunología , Hígado/inmunología , Isoformas de ProteínasRESUMEN
Hepatocyte nuclear factor-4α (Hnf4α) is a transcription factor that controls epithelial cell polarity and morphogenesis. Hnf4α conditional deletion during postnatal development has minor effects on intestinal epithelium integrity but promotes activation of the Wnt/ß-catenin pathway without causing tumorigenesis. Here, we show that Hnf4α does not act as a tumor-suppressor gene but is crucial in promoting gut tumorigenesis in mice. Polyp multiplicity in ApcMin mice lacking Hnf4α is suppressed compared with littermate ApcMin controls. Analysis of microarray gene expression profiles from mice lacking Hnf4α in the intestinal epithelium identifies novel functions of this transcription factor in targeting oxidoreductase-related genes involved in the regulation of reactive oxygen species (ROS) levels. This role is supported with the demonstration that HNF4α is functionally involved in the protection against spontaneous and 5-fluorouracil chemotherapy-induced production of ROS in colorectal cancer cell lines. Analysis of a colorectal cancer patient cohort establishes that HNF4α is significantly upregulated compared with adjacent normal epithelial resections. Several genes involved in ROS neutralization are also induced in correlation with HNF4A expression. Altogether, the findings point to the nuclear receptor HNF4α as a potential therapeutic target to eradicate aberrant epithelial cell resistance to ROS production during intestinal tumorigenesis.
Asunto(s)
Perfilación de la Expresión Génica , Factor Nuclear 4 del Hepatocito/metabolismo , Mucosa Intestinal/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Adenoma/genética , Adenoma/metabolismo , Adenoma/patología , Animales , Antimetabolitos Antineoplásicos/farmacología , Neoplasias del Colon/genética , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Femenino , Fluorouracilo/farmacología , Células HCT116 , Células HT29 , Factor Nuclear 4 del Hepatocito/genética , Humanos , Immunoblotting , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/patología , Neoplasias Intestinales/genética , Neoplasias Intestinales/metabolismo , Neoplasias Intestinales/patología , Pólipos Intestinales/genética , Pólipos Intestinales/metabolismo , Pólipos Intestinales/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Análisis de Secuencia por Matrices de Oligonucleótidos , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND: Hnf4alpha, an epithelial specific transcriptional regulator, is decreased in inflammatory bowel disease and protects against chemically-induced colitis in mice. However, the precise role of this factor in maintaining normal inflammatory homeostasis of the intestine remains unclear. The aim of this study was to evaluate the sole role of epithelial Hnf4alpha in the maintenance of gut inflammatory homeostasis in mice. METHODOLOGY/PRINCIPAL FINDINGS: We show here that specific epithelial deletion of Hnf4alpha in mice causes spontaneous chronic intestinal inflammation leading to focal areas of crypt dropout, increased cytokines and chemokines secretion, immune cell infiltrates and crypt hyperplasia. A gene profiling analysis in diseased Hnf4alpha null colon confirms profound genetic changes in cell death and proliferative behaviour related to cancer. Among the genes involved in the immune protection through epithelial barrier function, we identify the ion transporter claudin-15 to be down-modulated early in the colon of Hnf4alpha mutants. This coincides with a significant decrease of mucosal ion transport but not of barrier permeability in young animals prior to the manifestation of the disease. We confirm that claudin-15 is a direct Hnf4alpha gene target in the intestinal epithelial context and is down-modulated in mouse experimental colitis and inflammatory bowel disease. CONCLUSION: Our results highlight the critical role of Hnf4alpha to maintain intestinal inflammatory homeostasis during mouse adult life and uncover a novel function for Hnf4alpha in the regulation of claudin-15 expression. This establishes Hnf4alpha as a mediator of ion epithelial transport, an important process for the maintenance of gut inflammatory homeostasis.
Asunto(s)
Colitis/inducido químicamente , Colon/metabolismo , Factor Nuclear 4 del Hepatocito/metabolismo , Enfermedades Inflamatorias del Intestino/metabolismo , Transporte Iónico , Animales , Claudinas , Eliminación de Gen , Homeostasis , Inflamación , Mucosa Intestinal/metabolismo , Proteínas de la Membrana/biosíntesis , Ratones , Ratones Transgénicos , Análisis de Secuencia por Matrices de Oligonucleótidos , Transcripción GenéticaRESUMEN
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.