RESUMEN
Endosomal trafficking is intricately linked to G protein-coupled receptors (GPCR) fate and signaling. Extracellular uridine diphosphate (UDP) acts as a signaling molecule by selectively activating the GPCR P2Y6. Despite the recent interest for this receptor in pathologies, such as gastrointestinal and neurological diseases, there is sparse information on the endosomal trafficking of P2Y6 receptors in response to its endogenous agonist UDP and synthetic selective agonist 5-iodo-UDP (MRS2693). Confocal microscopy and cell surface ELISA revealed delayed internalization kinetics in response to MRS2693 vs. UDP stimulation in AD293 and HCT116 cells expressing human P2Y6. Interestingly, UDP induced clathrin-dependent P2Y6 internalization, whereas receptor stimulation by MRS2693 endocytosis appeared to be associated with a caveolin-dependent mechanism. Internalized P2Y6 was associated with Rab4, 5, and 7 positive vesicles independent of the agonist. We have measured a higher frequency of receptor expression co-occurrence with Rab11-vesicles, the trans-Golgi network, and lysosomes in response to MRS2693. Interestingly, a higher agonist concentration reversed the delayed P2Y6 internalization and recycling kinetics in the presence of MRS2693 stimulation without changing its caveolin-dependent internalization. This work showed a ligand-dependent effect affecting the P2Y6 receptor internalization and endosomal trafficking. These findings could guide the development of bias ligands that could influence P2Y6 signaling.
Asunto(s)
Receptores Acoplados a Proteínas G , Uridina Difosfato , Humanos , Ligandos , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Uridina Difosfato/metabolismo , Proteínas de Unión al GTP/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
In the intestine, mucins are expressed and secreted by goblet cells and enterocytes in a constitutive manner and in response to secretagogues to form a protective mucus layer. This protective barrier is often lost in inflammatory bowel disease (IBD). Interestingly, extracellular nucleotides, through P2Y receptors, were identified as mucin secretagogues in mucinous epithelia. These nucleotides are found in the intestine's extracellular milieu under basal conditions and in higher concentrations in pathologies such as IBD. It was observed that the mucus layer was affected in P2ry6 knockout mice suffering from dextran sodium sulfate (DSS)-induced colitis. P2ry6-/- mice were more sensitive to DSS-induced colitis, resulting in larger ulcers and increased disease activity index. Interestingly, the absence of P2Y6 receptor expression negatively affected the mucus quality, as shown by a reduction in sulfomucin staining and the absence of a dense internal fucosylated mucin layer in P2ry6-/- mice. Hence, we cannot rule out that the absence of P2Y6 receptors in knockout animals could negatively impact mucin secretion. However, we did not measure a reduction in the number of goblet cells, as previously reported. Instead, the results suggest that goblet cells rapidly discharged mucins to compensate for the mucus layer's increased lability, which resulted in empty goblet cells that are less visible to mucin staining. This study's results, along with previous reports, point toward a protective role for the P2Y6 receptor in IBD.
Asunto(s)
Colitis/genética , Enfermedades Inflamatorias del Intestino/genética , Mucinas/genética , Receptores Purinérgicos P2/genética , Animales , Colitis/inducido químicamente , Colitis/patología , Sulfato de Dextran/toxicidad , Regulación de la Expresión Génica/genética , Células Caliciformes/efectos de los fármacos , Humanos , Enfermedades Inflamatorias del Intestino/inducido químicamente , Enfermedades Inflamatorias del Intestino/patología , Mucosa Intestinal/efectos de los fármacos , Intestinos/efectos de los fármacos , Intestinos/patología , Ratones , Ratones Noqueados , Mucinas/farmacología , Moco/efectos de los fármacosRESUMEN
Bone mineralization is an orchestrated process by which mineral crystals are deposited by osteoblasts; however, the detailed mechanisms remain to be elucidated. The presence of P2X7 receptor (P2X7R) in immature and mature bone cells is well established, but contrasting evidence on its role in osteogenic differentiation and deposition of calcified bone matrix remains. To clarify these controversies in the present study, we investigated P2X7R participation in bone maturation. We demonstrated that the P2X7R is expressed and functional in human primary osteoblasts, and identified in the P2RX7 promoter several binding sites for transcription factors involved in bone mineralization. Of particular interest was the finding that P2X7R expression is enhanced by nuclear factor of activated T cells cytoplasmic 1 (NFATc1) overexpression, and accordingly, NFATc1 is recruited at the P2RX7 gene promoter in SaOS2 osteoblastic-like cells. In conclusion, our data provide further insights into the regulation of P2X7R expression and support the development of drugs targeting this receptor for the therapy of bone diseases.
Asunto(s)
Factores de Transcripción NFATC/genética , Factores de Transcripción NFATC/metabolismo , Osteoblastos/metabolismo , Receptores Purinérgicos P2X7/genética , Receptores Purinérgicos P2X7/metabolismo , Calcificación Fisiológica/genética , Diferenciación Celular/fisiología , Células Cultivadas , Femenino , Expresión Génica/genética , Humanos , Masculino , Persona de Mediana Edad , Osteocitos/metabolismo , Osteogénesis/genética , Regiones Promotoras Genéticas/genética , Transducción de Señal/genéticaRESUMEN
Cell migration is a ubiquitous process necessary to maintain and restore tissue functions. However, in cancer, cell migration leads to metastasis development and thus worsens the prognosis. Although the mechanism of cell migration is well understood, the identification of new targets modulating cell migration and deciphering their signaling events could lead to new therapies to restore tissue functions in diseases, such as inflammatory bowel disease, or to block metastatic development in different forms of cancer. Previous research has identified the G-protein-coupled P2Y6 receptor as an innovative target that could dictate cell migration under normal and pathological conditions. Surprisingly, there is little information on the cellular events triggered by activated P2Y6 during cell migration. Here, we demonstrated that P2Y6 activation stimulated A549 human lung cancer cells and Caco-2 colorectal cancer cell migration. Activated P2Y6 increased the number of filopodia and focal adhesions; two migratory structures required for cell migration. The generation of these structures involved Gαq /calcium/protein kinases C (PKC) and Gα13 /RHO-associated protein kinase-dependent pathways that dictate the formation of the migratory structures. These pathways led to the stabilization of the actin cytoskeleton through a PKC-dependent phosphorylation of cofilin. These results support the idea that the P2Y6 receptor represents a target of interest to modulate cell migration and revealed an intricate dialogue between two Gα-protein signaling pathways.
Asunto(s)
Movimiento Celular/genética , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/genética , Proteína Quinasa C-alfa/genética , Receptores Purinérgicos P2/genética , Células A549 , Actinas/genética , Células CACO-2 , Calcio/metabolismo , Extensiones de la Superficie Celular/genética , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Células Epiteliales/metabolismo , Subunidades alfa de la Proteína de Unión al GTP G12-G13/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Receptores Acoplados a Proteínas G/genética , Transducción de Señal/genética , Quinasas Asociadas a rho/genéticaRESUMEN
Inflammatory bowel disease (IBD) is a risk factor for the development of colorectal cancer (CRC) for which mutation to p53 is an early event leading to dysplasia. Interestingly, P2RY6 mRNA increases in both pathologies. In this study, we investigated if p53 and p53R273H mutant, commonly found in CRC and IBD, were involved in the transcriptional regulation of P2RY6. First, the P2RY6 promoter was defined as a region corresponding to -1600 to +273 nucleotides relative to the putative TATA-less transcriptional starting site found at position 73,264,505 of NCBI reference sequence NC_000010.11. We cloned this promoter region along with 5'-deletion constructs in the pGL4.10[luc2] vector for luciferase assays to delineate the minimal promoter region. We observed that p53 wt and p53R273H differentially regulated the transcription of the P2RY6 gene. In fact, increasing quantity of p53R273H enhanced the capacity of p53 wt to stimulate the transactivation of the P2RY6 promoter but this cooperative effect was lost when p53R273H was present in a ratio of 3:1. In accordance with the luciferase assays, ChIP analysis revealed that endogenous p53 wt was significantly associated with the P2RY6 proximal promoter, whereas the interaction of the p53R273H with the P2RY6 promoter was not significant. Although further studies are required to fully elucidate the molecular determinant controlling P2Y6 expression in diseases, we propose, for the first time, a molecular mechanism involving a collaboration between p53 wt and p53R273H to regulate the expression of this receptor.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Proteínas de Neoplasias/genética , Receptores Purinérgicos P2/genética , Transcripción Genética , Proteína p53 Supresora de Tumor/genética , Células A549 , Sustitución de Aminoácidos , Células CACO-2 , Proliferación Celular , Inmunoprecipitación de Cromatina , Genes Reporteros , Células HCT116 , Células HT29 , Humanos , Luciferasas/genética , Luciferasas/metabolismo , Proteínas de Neoplasias/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Receptores Purinérgicos P2/metabolismo , Transducción de Señal , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Overexpression of ecto-nucleotide pyrophosphatase-1 (NPP1) is associated with diseases such as calcium pyrophosphate dihydrate deposition disease, calcific aortic valve disease, and type 2 diabetes. In this context, NPP1 inhibitors are potential drug candidates for the treatment of these diseases. The present study focuses on the analysis of the structure-activity relationship of NPP1 inhibitors based on acyclic uracil-nucleotides. For this purpose, we synthesized acyclic uridine-monophosphate analogs, 10-11, uridine-diphosphate analogs, 12-14, and uridine-Pα,α-dithio-triphosphate analogs, 15-17. We evaluated their inhibitory activity and selectivity towards NPP1, -3, NTPDase1, -2, -3, and -8, and P2Y2,4,6 receptors. Analogs 16 and 17 were the most selective and potent NPP1 inhibitors (Ki 0.94 and 0.73⯵M, respectively) among the tested molecules. Analogs 10-17 had only minute effect on uracil-nucleotide responding P2Y2,4,6 receptors. Analog 17 (100⯵M) displayed 96% inhibition of NPPase activity in osteoarthritic human chondrocytes. Analogs 14-17 displayed weak inhibitory effect on alkaline phosphatase activity at equimolar concentrations in human chondrocytes. All tested analogs showed no toxicity at human chondrocytes. We concluded that ribose-ring to chain transformation, as well as the type of the nucleobase, are parameters of minor significance to NPP1 inhibition, whereas the major parameter is Pα-dithio-substitution. In addition, the length of the phosphate chain also significantly affects inhibition. Overall, the experimental results were well reproduced by molecular docking. A correlation was observed between the activities of the compounds and the number of H-bonds and salt bridges formed between the inhibitors and NPP1 binding site residues. Uracil-N1-(methoxy)ethyl-ß-Pα,α-dithio, Pß,γ-methylene tri-phosphate, 17, was identified as the most potent, selective, and non-toxic NPP1 inhibitor among the tested analogs, and may be used as a lead structure for further drug development.
Asunto(s)
Organofosfatos/farmacología , Inhibidores de Fosfodiesterasa/farmacología , Pirofosfatasas/antagonistas & inhibidores , Uracilo/farmacología , Condrocitos/efectos de los fármacos , Condrocitos/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Estructura Molecular , Organofosfatos/síntesis química , Organofosfatos/química , Inhibidores de Fosfodiesterasa/síntesis química , Inhibidores de Fosfodiesterasa/química , Hidrolasas Diéster Fosfóricas/metabolismo , Pirofosfatasas/metabolismo , Relación Estructura-Actividad , Uracilo/síntesis química , Uracilo/químicaRESUMEN
Extracellular nucleotides are important intercellular signaling molecules that were found enriched in the tumor microenvironment. In fact, interfering with G protein-coupled P2Y receptor signaling has emerged as a promising therapeutic alternative to treat aggressive and difficult-to-manage cancers such as those affecting the gastrointestinal system. In this review, we will discuss the functions of P2Y receptors in gastrointestinal cancers with an emphasis on colorectal, hepatic, and pancreatic cancers. We will show that P2Y2 receptor up-regulation increases cancer cell proliferation, tumor growth, and metastasis in almost all studied gastrointestinal cancers. In contrast, we will present P2Y6 receptor as having opposing roles in colorectal cancer vs. gastric cancer. In colorectal cancer, the P2Y6 receptor induces carcinogenesis by inhibiting apoptosis, whereas P2Y6 suppresses gastric cancer tumor growth by reducing ß-catenin transcriptional activity. The contribution of the P2Y11 receptor in the migration of liver and pancreatic cancer cells will be compared to its normal inhibitory function on this cellular process in ciliated cholangiocytes. Hence, we will demonstrate that the selective inhibition of the P2Y12 receptor activity in platelets was associated to a reduction in the risk of developing colorectal cancer and metastasis formation. We will succinctly review the role of P2Y1, P2Y4, P2Y13, and P2Y14 receptors as the knowledge for these receptors in gastrointestinal cancers is sparse. Finally, redundant ligand selectivity, nucleotide high lability, cell context, and antibody reliability will be presented as the main difficulties in defining P2Y receptor functions in gastrointestinal cancers.
Asunto(s)
Neoplasias Gastrointestinales/metabolismo , Receptores Purinérgicos P2Y/metabolismo , Humanos , Nucleótidos/metabolismo , Reproducibilidad de los Resultados , Microambiente Tumoral/fisiologíaRESUMEN
Ectonucleotide pyrophosphatase/phosphodiesterase-1 (NPP1) hydrolyzes phosphodiester bonds of nucleotides such as ATP, resulting mainly in the formation of AMP and pyrophosphate. NPP1 activity plays a deleterious function in calcified aortic valve disease and calcium pyrophosphate deposition disease. Thus, inhibitors of NPP1 represent a medical need. We developed novel NPP1 inhibitors based on uridine 5'-Pα,α-dithiophosphate analogues, 9-12. All these analogues potently inhibited hNPP1 (80-100% inhibition) at 100 µM, with no, or minimal, inhibition of NPP3 and other ectonucleotidases (NTPDase1,2,3,8). These compounds showed nearly no activity at uracil-nucleotide sensitive P2Y2,4,6-receptors and thus represent highly selective NPP1 inhibitors. The most promising inhibitor was diuridine 5'-Pα,α,5â³-Pα,α-tetrathiotetraphosphate, 12, exhibiting Ki of 27 nM. Analogues 9-12 proved to be highly stable to air oxidation and to acidic and basic pH. Docking simulations suggested that the enhanced NPP1 inhibitory activity and selectivity of analogue 12 could be attributed to the simultaneous occupancy of two sites (the AMP site and an alternative site) of NPP1 by this compound.
Asunto(s)
Diseño de Fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Pirofosfatasas/antagonistas & inhibidores , Nucleótidos de Uracilo/química , Nucleótidos de Uracilo/farmacología , Estabilidad de Medicamentos , Inhibidores Enzimáticos/metabolismo , Humanos , Hidrólisis , Concentración 50 Inhibidora , Simulación del Acoplamiento Molecular , Hidrolasas Diéster Fosfóricas/química , Hidrolasas Diéster Fosfóricas/metabolismo , Conformación Proteica , Pirofosfatasas/química , Pirofosfatasas/metabolismo , Relación Estructura-Actividad , Especificidad por Sustrato , Nucleótidos de Uracilo/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
In intestinal epithelial cells (IEC), it was reported that the activation of the P2X7 receptor leads to the internalization of the glucose transporter GLUT2, which is accompanied by a reduction of IEC capacity to transport glucose. In this study, we used P2rx7 -/- mice to decipher P2X7 functions in intestinal glucose transport and to evaluate the impacts on metabolism. Immunohistochemistry analyses revealed the presence of GLUT2 at the apical domain of P2rx7 -/- jejunum enterocytes. Positron emission tomography and biodistribution studies demonstrated that glucose was more efficiently delivered to the circulation of knockout animals. These findings correlated with increase blood glucose, insulin, triglycerides and cholesterol levels. In fact, P2rx7 -/- mice had increased serum triglyceride and cholesterol levels and displayed glucose intolerance and resistance to insulin. Finally, P2rx7 -/- mice developed a hepatic steatosis characterized by a reduction of Acaca, Acacb, Fasn and Acox1 mRNA expression, as well as for ACC and FAS protein expression. Our study suggests that P2X7 could play a central role in metabolic diseases.
Asunto(s)
Hígado Graso/metabolismo , Glucosa/metabolismo , Intestinos/química , Receptores Purinérgicos P2X7/deficiencia , Absorción Fisiológica , Animales , Transporte Biológico , Colesterol/metabolismo , Regulación hacia Abajo/genética , Dislipidemias/complicaciones , Dislipidemias/patología , Enterocitos/metabolismo , Fluorodesoxiglucosa F18/metabolismo , Transportador de Glucosa de Tipo 2/metabolismo , Hiperglucemia/complicaciones , Hiperglucemia/patología , Insulina/metabolismo , Resistencia a la Insulina , Yeyuno/metabolismo , Metabolismo de los Lípidos/genética , Masculino , Ratones , Receptores Purinérgicos P2X7/genética , Receptores Purinérgicos P2X7/metabolismo , Distribución Tisular , Triglicéridos/metabolismo , Aumento de PesoRESUMEN
Purinergic signaling has recently emerged as a network of signaling molecules, enzymes and receptors that coordinates the action and behavior of cancerous cells. Extracellular adenosine 5' triphosphate activates a plethora of P2 nucleotide receptors that can putatively modulate cancer cell proliferation, survival and dissemination. In this context, the G protein-coupled P2Y2 receptor was identified as one of the entities coordinating the cellular and molecular events that characterize cancerous cells. In this chapter, we will look at the contribution of the P2Y2 receptor in cancer outcomes and use this information to demonstrate that the P2Y2 receptor represents a drug target of interest in the setting of colorectal cancer, for which the role and function of this receptor is poorly defined. More particularly, we will review how the P2Y2 receptor modulates cancer cell proliferation and survival, while promoting cell dissemination and formation of metastases. Finally, we will investigate how the P2Y2 receptor can contribute to the detrimental development of drug resistance that is often observed in cancerous cells.
Asunto(s)
Proliferación Celular , Neoplasias Colorrectales/metabolismo , Resistencia a Antineoplásicos , Proteínas de Neoplasias/metabolismo , Receptores Purinérgicos P2Y2/metabolismo , Animales , Supervivencia Celular , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Humanos , Metástasis de la Neoplasia , Proteínas de Neoplasias/genética , Receptores Purinérgicos P2Y2/genéticaRESUMEN
The intestinal epithelial barrier is critical to limit potential harmful consequences from exposure to deleterious luminal contents on the organism. Although this barrier is functionally important along the entire gut, specific regional regulatory mechanisms involved in the maintenance of this barrier are poorly defined. Herein, we identified Gata4 as a crucial regulator of barrier integrity in the mouse proximal intestinal epithelium. Conditional deletion of Gata4 in the intestine led to a drastic increase in claudin-2 expression that was associated with an important increase of gut barrier permeability without causing overt spontaneous inflammation. Administration of indomethacin, a non-steroidal anti-inflammatory drug (NSAID) that causes enteritis, led to rapid and restricted proximal small intestinal injuries in Gata4 mutant mice as opposed to control mice. Comparative analysis of gene transcript profiles from indomethacin-challenged control and Gata4 mutant mice identified defects in epithelial cell survival, inflammatory cell recruitment and tissue repair mechanisms. Altogether, these observations identify Gata4 as a novel crucial regulator of the intestinal epithelial barrier and as a critical epithelial transcription factor implicated in the maintenance of proximal intestinal mucosal integrity after injury.
Asunto(s)
Enteritis/genética , Factor de Transcripción GATA4/genética , Indometacina/efectos adversos , Mucosa Intestinal/metabolismo , Animales , Claudinas/metabolismo , Enteritis/inducido químicamente , Enteritis/metabolismo , Factor de Transcripción GATA4/metabolismo , Regulación de la Expresión Génica , Ratones , Mutación , Salmonella typhiRESUMEN
P2Y6 receptor (P2Y6-R) is involved in various physiological and pathophysiological events. With a view to set rules for the design of UDP-based reversible P2Y6-R antagonists as potential drugs, we established structure-activity relationship of UDP analogues, bearing modifications at the uracil ring, ribose moiety, and the phosphate chain. For instance, C5-phenyl- or 3-NMe-uridine-5'-α,ß-methylene-diphosphonate, 16 and 23, or lack of 2'-OH, in 12-15, resulted in loss of both agonist and antagonist activity toward hP2Y6-R. However, uridylyl phosphosulfate, 19, selectively inhibited hP2Y6-R (IC50 112 µM) versus P2Y2/4-Rs. In summary, we have established a comprehensive SAR for hP2Y6-R ligands towards the development of hP2Y6-R antagonists.
Asunto(s)
Receptores Purinérgicos P2/química , Nucleótidos de Uracilo/síntesis química , Humanos , Estructura Molecular , Relación Estructura-Actividad , Nucleótidos de Uracilo/químicaRESUMEN
Extracellular adenosine 5'-triphosphate (ATP) is a signaling molecule that induces a plethora of effects ranging from the regulation of cell proliferation to modulation of cancerous cell behavior. In colorectal cancer, ATP was reported to stimulate epithelial cell proliferation and possibly promote resistance to anti-cancer treatments. However, the exact role of this danger-signaling molecule on cancerous intestinal epithelial cells (IECs) in response to chemotherapeutic agents remains unknown. To address how ATP may influence the response of cancerous IECs to chemotherapeutic agents, we used Caco-2 cells, which display enterocyte-like features, to determine the effect of ATP on the expression of multidrug resistance-associated protein 2 (MRP2). Gene and protein expression were determined by quantitative real-time PCR (qRT-PCR) and Western blotting. Resistance to etoposide, cisplatin and doxorubicin was determined by MTT assays in response to ATP stimulation of Caco-2 cells and in cells for which MRP2 expression was down-regulated by shRNA. ATP increased the expression of MRP2 at both the mRNA and protein levels. MRP2 expression involved an ATP-dependent stimulation of the MEK/ERK signaling pathway that was associated with an increase in relative resistance of Caco-2 cells to etoposide. Abolition of MRP2 expression using shRNA significantly reduced the protective effect of MRP2 toward etoposide as well as to cisplatin and doxorubicin. This study describes the mechanism by which ATP may contribute to the chemoresistance of cancerous IECs in colorectal cancer. Given the heterogeneity of colorectal adenocarcinoma responses to anti-cancer drugs, these findings call for further study to understand the role of P2 receptors in cancer drug therapy and to develop novel therapies aimed at regulating P2 receptor activity.
Asunto(s)
Adenosina Trifosfato/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Receptores Purinérgicos P2Y/genética , Antineoplásicos/farmacología , Células CACO-2 , Supervivencia Celular/efectos de los fármacos , Cisplatino/farmacología , Doxorrubicina/farmacología , Resistencia a Antineoplásicos/genética , Etopósido/farmacología , Células HEK293 , Humanos , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/antagonistas & inhibidores , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Receptores Purinérgicos P2Y/metabolismo , Transducción de Señal , Transcripción GenéticaRESUMEN
Activation of the ATP-dependent P2X7 receptor modulates glucose transport in intestinal epithelial cells through the downregulation of glucose transporter GLUT2. In the present study, we show that an increase in glucose concentration stimulates P2X7 receptor transcription via modulation of CCAAT/enhancer binding proteins (C/EBPs) α and ß expression. The described human P2X7 receptor promoter region (GenBank Y12851) was cloned upstream of a luciferase reporter gene in pGL4.10 plasmid and used to determine whether C/EBPs, namely C/EBPα and C/EBPß, are able to stimulate the transcription of P2X7 receptor. Results show that C/EBPß was the main regulator of P2X7 receptor expression in response to a glucose challenge. Chromatin immunoprecipitation (ChIP) assays further revealed that C/EBPß occupied the -213 to +6 nt P2X7 promoter region. Surprisingly, C/EBPα was also able to bind this region as revealed by ChIP assays, but without inducing receptor transcription. In fact, C/EBPα and the C/EBPß-LIP isoform blocked the C/EBPß-dependent regulation of P2X7 receptor transcription. These findings suggest that glucose is not only the major source of energy for cell function but may also act as a signaling molecule to stimulate the expression of regulatory proteins.
Asunto(s)
Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Proteínas de Unión al ADN/metabolismo , Células Epiteliales/metabolismo , Glucosa/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Animales , Sitios de Unión , Proteína beta Potenciadora de Unión a CCAAT/genética , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Proteínas de Unión al ADN/genética , Regulación hacia Abajo , Regulación de la Expresión Génica , Humanos , Intestinos/citología , Ratones , Ratones Noqueados , Regiones Promotoras Genéticas , Unión Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ratas , Receptores Purinérgicos P2X7/genéticaRESUMEN
The gastrointestinal tract is a passageway for dietary nutrients, microorganisms and xenobiotics. The gut is home to diverse bacterial communities forming the microbiota. While bacteria and their metabolites maintain gut homeostasis, the host uses innate and adaptive immune mechanisms to cope with the microbiota and luminal environment. In recent years, multiple bi-directional instructive mechanisms between microbiota, luminal content and mucosal immune systems have been uncovered. Indeed, epithelial and immune cell-derived mucosal signals shape microbiota composition, while microbiota and their by-products shape the mucosal immune system. Genetic and environmental perturbations alter gut mucosal responses which impact on microbial ecology structures. On the other hand, changes in microbiota alter intestinal mucosal responses. In this review, we discuss how intestinal epithelial Paneth and goblet cells interact with the microbiota, how environmental and genetic disorders are sensed by endoplasmic reticulum stress and autophagy responses, how specific bacteria, bacterial- and diet-derived products determine the function and activation of the mucosal immune system. We will also discuss the critical role of HDAC activity as a regulator of immune and epithelial cell homeostatic responses.
Asunto(s)
Homeostasis , Mucosa Intestinal/metabolismo , Animales , Estrés del Retículo Endoplásmico , Ambiente , Humanos , Mucosa Intestinal/citología , Mucosa Intestinal/inmunología , Mucosa Intestinal/microbiología , Microbiota , Receptores de Hidrocarburo de Aril/metabolismoRESUMEN
Histone deacetylases (Hdac) remove acetyl groups from proteins, influencing global and specific gene expression. Hdacs control inflammation, as shown by Hdac inhibitor-dependent protection from dextran sulfate sodium (DSS)-induced murine colitis. Although tissue-specific Hdac knockouts show redundant and specific functions, little is known of their intestinal epithelial cell (IEC) role. We have shown previously that dual Hdac1/Hdac2 IEC-specific loss disrupts cell proliferation and determination, with decreased secretory cell numbers and altered barrier function. We thus investigated how compound Hdac1/Hdac2 or Hdac2 IEC-specific deficiency alters the inflammatory response. Floxed Hdac1 and Hdac2 and villin-Cre mice were interbred. Compound Hdac1/Hdac2 IEC-deficient mice showed chronic basal inflammation, with increased basal disease activity index (DAI) and deregulated Reg gene colonic expression. DSS-treated dual Hdac1/Hdac2 IEC-deficient mice displayed increased DAI, histological score, intestinal permeability, and inflammatory gene expression. In contrast to double knockouts, Hdac2 IEC-specific loss did not affect IEC determination and growth, nor result in chronic inflammation. However, Hdac2 disruption protected against DSS colitis, as shown by decreased DAI, intestinal permeability and caspase-3 cleavage. Hdac2 IEC-specific deficient mice displayed increased expression of IEC gene subsets, such as colonic antimicrobial Reg3b and Reg3g mRNAs, and decreased expression of immune cell function-related genes. Our data show that Hdac1 and Hdac2 are essential IEC homeostasis regulators. IEC-specific Hdac1 and Hdac2 may act as epigenetic sensors and transmitters of environmental cues and regulate IEC-mediated mucosal homeostatic and inflammatory responses. Different levels of IEC Hdac activity may lead to positive or negative outcomes on intestinal homeostasis during inflammation.
Asunto(s)
Colitis/enzimología , Colon/enzimología , Células Epiteliales/enzimología , Histona Desacetilasa 1/metabolismo , Histona Desacetilasa 2/metabolismo , Mucosa Intestinal/enzimología , Animales , Colitis/genética , Colitis/inmunología , Colitis/patología , Colon/inmunología , Colon/patología , Sulfato de Dextran , Modelos Animales de Enfermedad , Epigénesis Genética , Células Epiteliales/inmunología , Células Epiteliales/patología , Regulación de la Expresión Génica , Genotipo , Histona Desacetilasa 1/deficiencia , Histona Desacetilasa 1/genética , Histona Desacetilasa 2/deficiencia , Histona Desacetilasa 2/genética , Homeostasis , Inmunidad Mucosa , Mediadores de Inflamación/metabolismo , Mucosa Intestinal/inmunología , Mucosa Intestinal/patología , Ratones , Ratones Noqueados , Permeabilidad , Fenotipo , Factores de TiempoRESUMEN
C. difficile is a Gram-positive spore-forming anaerobic bacterium that is the leading cause of nosocomial diarrhea in the developed world. The pathogenesis of C. difficile infections (CDI) is driven by toxin A (TcdA) and toxin B (TcdB), secreted factors that trigger the release of inflammatory mediators and contribute to disruption of the intestinal epithelial barrier. Neutrophils play a key role in the inflammatory response and the induction of pseudomembranous colitis in CDI. TcdA and TcdB alter cytoskeletal signaling and trigger the release of CXCL8/IL-8, a potent neutrophil chemoattractant, from intestinal epithelial cells; however, little is known about the surface receptor(s) that mediate these events. In the current study, we sought to assess whether toxin-induced CXCL8/IL-8 release and barrier dysfunction are driven by the activation of the P2Y6 receptor following the release of UDP, a danger signal, from intoxicated Caco-2 cells. Caco-2 cells express a functional P2Y6 receptor and release measurable amounts of UDP upon exposure to TcdA/B. Toxin-induced CXCL8/IL-8 production and release were attenuated in the presence of a selective P2Y6 inhibitor (MRS2578). This was associated with inhibition of TcdA/B-induced activation of NFκB. Blockade of the P2Y6 receptor also attenuated toxin-induced barrier dysfunction in polarized Caco-2 cells. Lastly, pretreating mice with the P2Y6 receptor antagonists (MSR2578) attenuated TcdA/B-induced inflammation and intestinal permeability in an intrarectal toxin exposure model. Taken together these data outline a novel role for the P2Y6 receptor in the induction of CXCL8/IL-8 production and barrier dysfunction in response to C. difficile toxin exposure and may provide a new therapeutic target for the treatment of CDI.
Asunto(s)
Clostridioides difficile/metabolismo , Enterocolitis Seudomembranosa/metabolismo , Enterocolitis Seudomembranosa/fisiopatología , Enterotoxinas/metabolismo , Interleucina-8/biosíntesis , Mucosa Intestinal/metabolismo , Mucosa Intestinal/fisiopatología , Receptores Purinérgicos P2/metabolismo , Animales , Apirasa/metabolismo , Células CACO-2 , Modelos Animales de Enfermedad , Enterocolitis Seudomembranosa/genética , Humanos , Inflamación/genética , Inflamación/metabolismo , Mucosa Intestinal/microbiología , Masculino , Ratones , FN-kappa B/metabolismo , Antagonistas del Receptor Purinérgico P2/farmacología , Transducción de SeñalRESUMEN
With the diabetes epidemic affecting the world population, there is an increasing demand for means to regulate glycemia. Dietary glucose is first absorbed by the intestine before entering the blood stream. Thus, the regulation of glucose absorption by intestinal epithelial cells (IECs) could represent a way to regulate glycemia. Among the molecules involved in glycemia homeostasis, extracellular ATP, a paracrine signaling molecule, was reported to induce insulin secretion from pancreatic ß cells by activating P2Y and P2X receptors. In rat's jejunum, P2X7 expression was previously immunolocalized to the apex of villi, where it has been suspected to play a role in apoptosis. However, using an antibody recognizing the receptor extracellular domain and thus most of the P2X7 isoforms, we showed that expression of this receptor is apparent in the top two-thirds of villi. These data suggest a different role for this receptor in IECs. Using the non-cancerous IEC-6 cells and differentiated Caco-2 cells, glucose transport was reduced by more than 30% following P2X7 stimulation. This effect on glucose transport was not due to P2X7-induced cell apoptosis, but rather was the consequence of glucose transporter 2 (Glut2)'s internalization. The signaling pathway leading to P2X7-dependent Glut2 internalization involved the calcium-independent activation of phospholipase Cγ1 (PLCγ1), PKCδ, and PKD1. Although the complete mechanism regulating Glut2 internalization following P2X7 activation is not fully understood, modulation of P2X7 receptor activation could represent an interesting approach to regulate intestinal glucose absorption.