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
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
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
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
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
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.
Asunto(s)
Enterocitos/metabolismo , Transportador de Glucosa de Tipo 2/metabolismo , Agonistas del Receptor Purinérgico P2X/farmacología , Receptores Purinérgicos P2X7/metabolismo , Animales , Línea Celular , Regulación hacia Abajo , Glucosa/metabolismo , Transportador de Glucosa de Tipo 2/genética , Humanos , Proteína Quinasa C/metabolismo , Proteína Quinasa C-delta/metabolismo , Interferencia de ARN , ARN Interferente Pequeño , Ratas , Receptores Purinérgicos P2X7/genéticaRESUMEN
P2Y(2) receptor expression is increased in intestinal epithelial cells (IECs) during inflammatory bowel diseases (IBDs). In this context, P2Y(2) stimulates PGE(2) release by IECs, suggesting a role in wound healing. For this study, we have used the non-cancerous IEC-6 cell line. IEC-6 cell migration was determined using Boyden chambers and the single-edged razor blade model of wounding. The receptor was activated using ATP, UTP, or 2-thioUTP. Pharmacological inhibitors, a blocking peptide, a neutralizing antibody and interfering RNAs were used to characterize the signaling events. Focal adhesions and microtubule (MT) dynamics were determined by immunofluorescence using anti-vinculin and anti-acetylated-α-tubulin antibodies, respectively. In vivo, the dextran sodium sulfate mouse model of colitis was used to characterize the effects of P2Y(2) agonist 2-thioUTP on remission. We showed that P2Y(2) increased cell migration and wound closure by recruiting Go protein with the cooperation of integrin α(v) . Following P2Y(2) activation, we demonstrated that GSK3ß activity was inhibited in response to Akt activation. This leads to MT stabilization and increased number of focal adhesions. In vivo, P2Y(2) activation stimulates remission, as illustrated by a reduction in the disease activity index values and histological scores as compared to control mice. These findings highlight a novel function for this receptor in IECs. They also illustrate that P2Y receptors could be targeted for the development of innovative therapies for the treatment of IBDs.
Asunto(s)
Colitis/metabolismo , Células Epiteliales/metabolismo , Mucosa Intestinal/citología , Mucosa Intestinal/metabolismo , Microtúbulos/metabolismo , Receptores Purinérgicos P2Y2/metabolismo , Adenosina Trifosfato/farmacología , Animales , Calcio/metabolismo , Línea Celular , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Colitis/inducido químicamente , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Integrina alfaV/genética , Integrina alfaV/metabolismo , Mucosa Intestinal/efectos de los fármacos , Ratones , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Agonistas del Receptor Purinérgico P2Y/farmacología , Ratas , Receptores Purinérgicos P2Y2/genética , Tubulina (Proteína)/genética , Tubulina (Proteína)/inmunología , Tubulina (Proteína)/metabolismo , Uridina Trifosfato/farmacología , Cicatrización de Heridas/efectos de los fármacosRESUMEN
P2Y(6) nucleotide receptor (P2Y(6)-R) plays important physiological roles, such as insulin secretion and reduction of intraocular pressure. However, this receptor is still lacking potent and selective agonists to be used as potential drugs. Here, we synthesized uracil nucleotides and dinucleotides, substituted at the C5 and/or P(α) position with methoxy and/or borano groups, 18-22. Compound 18A, R(p) isomer of 5-OMe-UDP(α-B), is the most potent and P2Y(6)-R selective agonist currently known (EC(50) 0.008 µM) being 19-fold more potent than UDP and showing no activity at uridine nucleotide receptors, P2Y(2)- and P2Y(4)-R. Analogue 18A was highly chemically stable under conditions mimicking gastric juice acidity (t(1/2) = 16.9 h). It was more stable to hydrolysis by nucleotide pyrophosphatases (NPP1,3) than UDP (15% and 28% hydrolysis by NPP1 and NPP3, respectively, vs 50% and 51% hydrolysis of UDP) and metabolically stable in blood serum (t(1/2) = 17 vs 2.4, 11.9, and 21 h for UDP, 5-OMe-UDP, and UDP(α-B), respectively). This newly discovered highly potent and physiologically stable P2Y(6)-R agonist may be of future therapeutic potential.
Asunto(s)
Receptores Purinérgicos P2/metabolismo , Uridina Trifosfato/farmacología , Relación Dosis-Respuesta a Droga , Humanos , Modelos Moleculares , Estructura Molecular , Relación Estructura-Actividad , Uridina Trifosfato/síntesis química , Uridina Trifosfato/químicaRESUMEN
BACKGROUND: Inflammatory bowel diseases are characterized by the presence of CXCL8 at the site of lesions resulting in neutrophil recruitment and loss of tissue functions. We report that P2Y(6) receptor activation stimulates CXCL8 expression and release by intestinal epithelial cells (IECs). In this context, we investigated if uridine 5'-diphosphate (UDP) enemas stimulate neutrophil recruitment to the mucosa of mice suffering from colitis-like disease and we characterized the signaling events linking P2Y(6) to CXCL8 expression in IEC. METHODS: Neutrophil recruitment was monitored by immunofluorescence and FACS analysis. Expression of Cxcl1, a mouse functional homolog of CXCL8, was determined by quantitative real-time polymerase chain reaction (qPCR). Pharmacological inhibitors and interfering RNAs were used to characterize the signaling pathway. The outcomes of these treatments on protein phosphorylation and on CXCL8 expression were characterized by western blots, qPCR, luciferase, and chromatin immunoprecipitation (ChIP) assays. RESULTS: Mutation of the AP-1 site in the CXCL8 core promoter abolished the UDP-stimulating effect. The c-fos/c-jun dimer was identified as the AP-1 complex regulating CXCL8 in response to UDP stimulation. Regulation of CXCL8 expression by P2Y(6) required PKCδ activation upstream of the signaling pathway composed of MEK1/2-ERK1/2 and c-fos. UDP administration to mice suffering from colitis-like disease increased the number of neutrophil infiltrating the mucosa, correlating with Cxcl1 increased expression in IEC and the severity of inflammation. CONCLUSIONS: This study not only describes the P2Y(6) signaling mechanism regulating CXCL8 expression in IEC, but it also illustrates the potential of targeting P2Y(6) to reduce intestinal inflammation.
Asunto(s)
Células Epiteliales/inmunología , Inflamación/inmunología , Enfermedades Inflamatorias del Intestino/inmunología , Interleucina-8/genética , Mucosa Intestinal/inmunología , Infiltración Neutrófila , Receptores Purinérgicos P2/metabolismo , Factor de Transcripción AP-1/metabolismo , Animales , Western Blotting , Células Cultivadas , Quimiocina CXCL1/genética , Quimiocina CXCL1/metabolismo , Inmunoprecipitación de Cromatina , Células Epiteliales/metabolismo , Células Epiteliales/patología , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Inflamación/metabolismo , Inflamación/patología , Enfermedades Inflamatorias del Intestino/metabolismo , Enfermedades Inflamatorias del Intestino/patología , Interleucina-8/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Luciferasas/metabolismo , Ratones , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fosforilación , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores Purinérgicos P2/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Factor de Transcripción AP-1/genéticaRESUMEN
BACKGROUND/AIMS: The inositol 1,4,5-trisphosphate receptor (IP(3)R), a ligand-gated Ca(2+) channel, plays an important role in the control of intracellular Ca(2+). Three isoforms of IP(3)R have been identified and most cell types express different proportions of these isoforms. The purpose of this study was to investigate how IP(3)R signalling is involved in the activation of the Ca(2+)-sensitive transcription factors NFAT and CREB. METHODS: Each IP(3)R isoform expressed in HEK 293A cells was knocked down using selective siRNA. Free intracellular Ca(2+) was monitored spectrofluometrically. NFAT and CREB activities were measured with luciferase reporter constructs. RESULTS: IP(3)R-2-knocked down HEK 293A cells showed a deficient CCh-induced Ca(2+) response that could be rescued by co-stimulation with VIP, a cAMP increasing agonist. NFAT transcriptional activity, but not CREB transcriptional activity, was significantly reduced in IP(3)R-2-knocked down HEK 293A cells. Overexpression of IP(3)R-1 could fully compensate for IP(3)R-2 knock down to mobilize Ca(2+) and to activate NFAT. CONCLUSION: Our results show that the knock down of IP(3)R-2 significantly reduced the intracellular Ca(2+) response of HEK 293 cells. This reduced Ca(2+) response did not affect the activation of CREB but significantly decreased the activation of NFAT, suggesting that the Ca(2+) signals required for the activation of NFAT are stronger than those required for the activation of CREB.
Asunto(s)
Calcio/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Factores de Transcripción NFATC/metabolismo , Células HEK293 , Humanos , Receptores de Inositol 1,4,5-Trifosfato/genética , Interferencia de ARN , ARN Interferente PequeñoRESUMEN
BACKGROUND: Ascorbic acid is an essential vitamin and a powerful antioxidant. Many studies have highlighted the benefits of ascorbic acid for chronic cardiovascular diseases such as hypertension in which angiotensin II (Ang II) plays an significant role. We therefore hypothesized that ascorbic acid could modify the pharmacological properties of the AT(1) receptor for Ang II. METHODS: Binding studies and Ca(2+) mobilization studies were performed with HEK293 cells stably expressing the AT(1) receptor for Ang II. Smooth muscle contraction studies were performed with rabbit aorta strips that endogenously express the AT(1) receptor. RESULTS: Scatchard analysis revealed that ascorbic acid decreased the binding affinity of the AT(1) receptor without modifying its maximal binding capacity. Ascorbic acid did not modify the binding affinity of the AT(2) receptor for Ang II or of the UT receptor for urotensin II. In single-cell Ca(2+) imaging assays, ascorbic acid reduced the frequency of intracellular Ca(2+) oscillations induced by a low dose of Ang II. In functional assays, ascorbic acid significantly diminished the contraction of rabbit aorta pre-contracted with Ang II but not those pre-contracted with urotensin II. CONCLUSIONS: Ascorbic acid decreases the binding affinity of the AT(1) receptor. These results offer a mechanistic explanation for the reported blood pressure lowering effect of ascorbic acid.
Asunto(s)
Angiotensina II/metabolismo , Antihipertensivos/farmacología , Ácido Ascórbico/farmacología , Señalización del Calcio/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Receptor de Angiotensina Tipo 1/efectos de los fármacos , Vasoconstricción/efectos de los fármacos , Animales , Línea Celular , Relación Dosis-Respuesta a Droga , Humanos , Músculo Liso Vascular/metabolismo , Conejos , Receptor de Angiotensina Tipo 1/genética , Receptor de Angiotensina Tipo 1/metabolismo , Factores de Tiempo , TransfecciónRESUMEN
In non-excitable cells, the inositol 1,4,5-trisphosphate receptor channel, which plays a major (IP(3)R) is an intracellular Ca(2+) role in Ca(2+) signalling. Three isoforms of IP(3)R have been identified (IP(3)R-1, IP(3)R-2 and IP(3)R-3) and most cell types express different proportions of each isoform. The differences between the pharmacological and functional properties of the various isoforms of IP(3)R are poorly understood. AR4-2J cells, which express almost exclusively (~86%) the IP(3)R-2, represent an interesting model to study this particular isoform. Here, we investigated a regulatory mechanism by which protein kinase C (PKC) influences IP(3)R-2-mediated Ca(2+) release. Using an immunoprecipitation approach, we confirmed that AR4-2J cells express almost exclusively the IP(3)R-2 isoform. Using an in vitro phosphorylation assay, we showed that the immunopurified IP(3)R-2 was efficiently phosphorylated by exogenous PKC. In intact AR4-2J cells metabolically labelled with (32)Pi, we showed that phorbol-12-myristate-13-acetate (PMA) and Ca(2+) mobilizing agonists cause the phosphorylation of IP(3)R-2. In saponin-permeabilized AR4-2J cells, IP(3)-induced Ca(2+) release was reduced after a pre-treatment with PMA or with exogenous PKC. PMA also reduced the Ca(2+) response of intact AR4-2J cells stimulated with carbachol and epidermal growth factor, two agonists that use different receptor types to activate phospholipase C. These results demonstrate that PKC decreases the Ca(2+)mobilizing activity of IP(3)R-2 and thus exerts a negative feedback on the agonists-induced Ca(2+) response of AR4-2J cells.
Asunto(s)
Canales de Calcio/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Páncreas/metabolismo , Proteína Quinasa C/metabolismo , Animales , Transporte Biológico , Calcio/análisis , Calcio/metabolismo , Línea Celular Tumoral , Citosol/química , Citosol/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Inositol 1,4,5-Trifosfato/farmacología , Ratas , Espectrometría de FluorescenciaRESUMEN
In non-excitable cells, the inositol 1,4,5-trisphosphate receptor (IP3R) is an intracellular Ca2+ channel which plays a major role in Ca2+ signalling. Three isoforms of IP3R have been identified (IP3R-1, IP3R-2 and IP3R-3) and most cell types express different proportions of each isoform. The differences between the pharmacological and functional properties of the various isoforms of IP3R are poorly known. RINm5F cells who express almost exclusively (approximately 90%) the IP3R-3, represent an interesting model to study this particular isoform. Here, we investigated a regulatory mechanism by which protein kinase C (PKC) may influence IP3R-3-mediated Ca2+ release. With an immunoprecipitation approach we confirmed that RINm5F cells express almost exclusively the IP3R-3 isoform. With an in vitro phosphorylation approach, we showed that the immunopurified IP3R-3 was efficiently phosphorylated by exogenous PKC. With a direct in cellulo approach and an indirect in cellulo back-phosphorylation approach we showed that phorbol-12-myristate-13-acetate (PMA) causes the phosphorylation of IP3R-3 in intact RINm5F cells. In saponin-permeabilized RINm5F cells, 3-induced Ca2+ release was reduced after a pre-treatment with PMA. PMA also reduced the Ca2+ response of intact RINm5F cells stimulated with carbachol and EGF, two agonists that use different receptor types to activate phospholipase C. These results suggest the existence of a negative feedback mechanism involving two components of the Ca2+ signalling cascade, whereby activated PKC dampens IP3R-3 activity.