RESUMEN
Head and neck squamous cell carcinomas (HNSCCs) are a heterogenous group of tumors and among the top 10 most common cancers and they arise from the epithelial tissues of the mucosal surfaces of the oral cavity, oropharynx, and larynx. Aberrant purinergic signaling has been associated with various cancer types. Here, we studied the role of the P2Y2 purinergic receptor (P2Y2R) in the context of oral cancer. We utilized bioinformatics analysis of deposited datasets to examine purinome gene expression in HNSCC tumors and cells lines and functionally characterized nucleotide-induced P2 receptor signaling in human FaDu and Cal27 and murine MOC2 oral cancer cell lines. Utilizing tumorigenesis assays with wild-type or P2ry2 knockout MOC2 cells we evaluated the role of P2Y2Rs in tumor growth and the host anti-tumor immune responses. Our data demonstrate that human and murine oral cancer cell lines express numerous P2 receptors, with the P2Y2R being highly expressed. Using syngeneic tumor grafts in wild-type mice, we observed that MOC2 tumors expressing P2Y2R were larger than P2Y2R-/- tumors. Wild-type MOC2 tumors contained a lower population of tumor-infiltrating CD11b+F4/80+ macrophages and CD3+ cells, which were revealed to be CD3+CD4+IFNγ+ T cells, compared to P2Y2R-/- tumors. These results were mirrored when utilizing P2Y2R-/- mice, indicating that the changes in MOC2 tumor growth and to the host anti-tumor immune response were independent of host derived P2Y2Rs. Results suggest that targeted suppression of the P2Y2R in HNSCC cells in vivo, rather than systemic P2Y2R antagonism, may be a more effective treatment strategy for HNSCCs.
RESUMEN
Hypertension has become a prominent public health concern. Essential hypertension (EH) is a polygenic disorder caused by multiple susceptibility genes. It has been previously shown that the purinergic P2Y2 receptor (P2Y2R) regulates blood pressure; however, whether P2Y2R genetic polymorphisms correlate with EH has not been investigated in Chinese. Our study included 500 EH cases and 504 controls who are Chinese postmenopausal women. We used allele-specific polymerase chain reaction (ASPCR) to genotype five single-nucleotide polymorphism (SNPs) in the P2Y2R gene, i.e., rs4944831, rs12366239, rs1783596, rs4382936, and rs10898909. We assessed the association of P2Y2R genetic polymorphisms with EH susceptibility. The results demonstrated that P2Y2R rs4382936A was correlated with a high risk of EH; particularly, the participants with the rs4382936A allele and CA/AA/(CA+AA) genotypes were at higher risks to EH compared to the subjects with the rs4382936C allele and CC genotype. Moreover, haplotype CAG combined by rs1783596-rs4382936-rs10898909 was a susceptible haplotype for EH, whereas haplotype CCG was a protective haplotype for EH. These results may provide new evidence for applying P2Y2R genetic polymorphisms as useful markers in clinic screening or monitoring potential EH cases in a population of Chinese postmenopausal women.
Asunto(s)
Hipertensión , Posmenopausia , Humanos , Femenino , Posmenopausia/genética , Hipertensión Esencial , Hipertensión/genética , Genotipo , Haplotipos , Polimorfismo de Nucleótido Simple/genética , China/epidemiología , Predisposición Genética a la Enfermedad/genética , Frecuencia de los GenesRESUMEN
Non-alcoholic fatty liver disease (NAFLD) is a chronic metabolic liver disease associated with obesity and insulin resistance. Activation of the purinergic receptor P2Y2R has been reported to promote adipogenesis, inflammation and dyslipidemia in adipose tissues in obese mice. However, the role of P2Y2R and its mechanisms in NAFLD remain unknown. We hypothesized that P2Y2R deficiency may play a protective role in NAFLD by modulating lipid metabolism in the liver. In this study, we fed wild type and P2Y2R knockout mice with a high-fat diet (HFD) for 12 weeks and analyzed metabolic phenotypes. First, P2Y2R deficiency effectively improved insulin resistance with a reduction in body weight and plasma insulin. Second, P2Y2R deficiency attenuated hepatic lipid accumulation and injury with reduced alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels. Third, P2Y2R deficiency decreased the expression of fatty acid synthesis mediators (cluster of differentiation (CD36), fatty acid synthase (FAS), and stearoyl-CoA desaturase 1 (SCD1)); and increased the expression of adipose triglyceride lipase (ATGL), a lipolytic enzyme. Mechanistically, P2Y2R deficiency increased the AMP-activated protein kinase (AMPK) activity to improve mitochondrial fatty acid ß-oxidation (FAO) by regulating acetyl-CoA carboxylase (ACC) and carnitine palmitoyltransferase 1A (CPT1A)-mediated FAO pathway. In addition, P2Y2R deficiency increased peroxisome proliferator-activated gamma co-activator-1α (PGC-1α)-mediated mitochondrial biogenesis. Conclusively, P2Y2R deficiency ameliorated HFD-induced hepatic steatosis by enhancing FAO through AMPK signaling and PGC-1α pathway, suggesting P2Y2R as a promising therapeutic target for NAFLD.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Ácidos Grasos/metabolismo , Lipogénesis/genética , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Receptores Purinérgicos P2Y2/metabolismo , Acetil-CoA Carboxilasa/metabolismo , Alanina Transaminasa/metabolismo , Animales , Aspartato Aminotransferasas/metabolismo , Peso Corporal , Antígenos CD36/metabolismo , Carnitina O-Palmitoiltransferasa/metabolismo , Dieta Alta en Grasa , Ácido Graso Sintasas/metabolismo , Insulina/sangre , Resistencia a la Insulina/fisiología , Lipasa/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Obesos , Mitocondrias/metabolismo , Enfermedad del Hígado Graso no Alcohólico/enzimología , Obesidad/metabolismo , Receptores Purinérgicos P2Y2/deficiencia , Receptores Purinérgicos P2Y2/genética , Estearoil-CoA Desaturasa/metabolismoRESUMEN
Purine signaling pathway plays an important role in inflammation and tissue damage. To investigate the role of purine signaling pathway in acute alcoholic liver injury and chronic alcoholic liver fibrosis, we replicated two animal models and two cellular models. We found that body weights, liver indexes, serum biochemical parameters, serum fibrosis indexes, and pathological and immunohistochemical results had significant changes in two treatment groups compared with two control groups. In addition, gene expressions of purine receptors, inflammatory cytokines, fibrogenic cytokines, and inflammasomes increased obviously in two animal models and two cellular models. Furthermore, purine receptor inhibitors could significantly inhibit protein expressions of purine receptors and reduce protein expressions of inflammatory cytokines, fibrogenic cytokines, and inflammasomes. Besides, P2X7R small interfering ribonucleic acid (siRNA) had the same effects. Meanwhile, we detected protein expressions of inflammatory cytokines secreted by inflammasomes, and we found that purine receptor-mediated inflammasomes activation was a key event in the process of chronic alcoholic liver fibrosis. In summary, this study shows that inhibition of purine receptors can alleviate acute alcoholic liver injury and chronic alcoholic liver fibrosis in mice. Therefore, purine receptor is a potential new target for the treatment of acute alcoholic liver injury and chronic alcoholic fibrosis.
Asunto(s)
Proliferación Celular , Citocinas/metabolismo , Células Estrelladas Hepáticas/metabolismo , Hepatopatías Alcohólicas/metabolismo , Purinas/metabolismo , Transducción de Señal , Animales , Células Estrelladas Hepáticas/patología , Inflamación/metabolismo , Inflamación/patología , Hepatopatías Alcohólicas/patología , Ratones , Receptores Purinérgicos P2X7/metabolismoRESUMEN
The disturbances of cellular proteostasis caused by the alteration in the ubiquitin-proteasome system (UPS) have been proposed as a common mechanism underlying several neural pathologies that involve a neuroinflammatory process. As we have previously reported that the nucleotide receptor P2Y purinoceptor 2 (P2Y2R) regulates the proteasomal catalytic activities, we wonder whether this receptor is involved in the UPS disturbances associated with the neuroinflammation process. With the use of mice expressing a UPS reporter [mice expressing the UPS reporter ubiquitinG76V-green fluorescent protein (UbGFP mice)], we found that LPS-induced acute neuroinflammation status causes a UPS impairment in astrocytes and microglial cells by a mechanism dependent on P2Y2R. In this line, LPS-treated double transgenic UbGFP; P2Y2R-/- mice did not present a UPS impairment in astrocytes or a social interaction deficit as severe as that observed in LPS-treated UbGFP mice. In vivo administration of selective P2Y2R agonist diuridine tetraphosphate reversed the UPS impairment completely in astrocytes and partially in microglial cells, promoting increased expression of the proteasomal ß5 subunit by a mechanism dependent on the Src/PI3K/ERK pathway. Altogether, our results suggest that LPS induces unbalanced proteostasis in astrocytes by blocking P2Y2R. Finally, our findings point to the design of selective P2Y2R agonist drugs as a new therapeutic approach to treat the neuroinflammatory status.-De Diego García, L., Sebastián-Serrano, Á., Hernández, I. H., Pintor, J., Lucas, J. J., Díaz-Hernández, M. The regulation of proteostasis in glial cells by nucleotide receptors is key in acute neuroinflammation.
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Astrocitos/metabolismo , Sistema de Señalización de MAP Quinasas , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteostasis , Receptores Purinérgicos P2Y2/metabolismo , Ubiquitina/metabolismo , Animales , Inflamación/inducido químicamente , Inflamación/tratamiento farmacológico , Inflamación/genética , Inflamación/metabolismo , Lipopolisacáridos/toxicidad , Ratones , Ratones Noqueados , Complejo de la Endopetidasa Proteasomal/genética , Receptores Purinérgicos P2Y2/genética , Conducta Social , Ubiquitina/genéticaRESUMEN
Several G-protein-coupled receptors (GPCRs) can be activated or inhibited in a specific manner by membrane-permeable pepducins, which are short palmitoylated peptides with amino acid sequences identical to an intracellular domain of the receptor to be targeted. Unlike the endogenous P2Y2R agonist ATP, the P2Y2PalIC2 pepducin, which has an amino acid sequence corresponding to the second intracellular loop of the human ATP receptor (P2Y2R), activated the superoxide anion-generating NADPH-oxidase in neutrophils. In addition to having a direct effect on neutrophils, the P2Y2R pepducin converted naïve neutrophils to a primed state, which secondarily responded to ATP by producing superoxide. A pepducin with a peptide identical to the third intracellular loop of P2Y2R (P2Y2PalIC3) exhibited the same basic functions as P2Y2PalIC2, whereas one with a peptide that was identical to the first intracellular loop (P2Y2PalIC1) lacked these functions. The responses induced in neutrophils by the P2Y2R pepducins were not inhibited by the P2Y2R antagonist AR-C118925, and the receptor desensitization profile suggested the involvement of FPR2 rather than P2Y2R. Accordingly, antagonists/inhibitors of FPR2 attenuated the activities of the P2Y2R pepducins, which also selectively activated FPR2-overexpressing cells. In summary, we show that pepducins supposed to target P2Y2R activate human neutrophils through FPR2. We also show that the P2Y2PalIC2 pepducin can convert ATP from a non-activating agent to a potent neutrophil NADPH-oxidase activator. The molecular basis of this phenomenon involves cross-talk between the receptor/ligand pairs of P2Y2R/ATP and FPR2/P2Y2-pepducin.
Asunto(s)
Adenosina Trifosfato/metabolismo , NADPH Oxidasas/metabolismo , Neutrófilos/metabolismo , Péptidos/metabolismo , Receptores de Formil Péptido/metabolismo , Receptores de Lipoxina/metabolismo , Receptores Purinérgicos P2Y2/metabolismo , Secuencia de Aminoácidos , Unión Competitiva/efectos de los fármacos , Calcio/metabolismo , Células Cultivadas , Células HL-60 , Humanos , Ligandos , Datos de Secuencia Molecular , Neutrófilos/citología , Neutrófilos/efectos de los fármacos , Oxígeno/metabolismo , Péptidos/farmacología , Unión Proteica/efectos de los fármacos , Receptor Cross-Talk/efectos de los fármacos , Receptores Purinérgicos P2Y2/química , Factores de TiempoRESUMEN
Epithelial tubular structures are essential units in various organs. Here, we used rat intestinal epithelial IEC6 cells to investigate tubulogenesis and we found that tubular formation was induced by a combination of Wnt3a and EGF signaling during three-dimensional culture. Wnt3a and EGF induced the expression of the P2Y2 receptor (P2Y2R, also known as P2RY2), and knockdown of P2Y2R suppressed tubular formation. A P2Y2R mutant that lacks nucleotide responsiveness rescued the phenotypes resulting from P2Y2R knockdown, suggesting that nucleotide-dependent responses are not required for P2Y2R functions in tubular formation. The Arg-Gly-Asp (RGD) sequence of P2Y2R has been shown to interact with integrins, and a P2Y2R mutant lacking integrin-binding activity was unable to induce tubular formation. P2Y2R expression inhibited the interaction between integrins and fibronectin, and induced cell morphological changes and proliferation. Inhibition of integrin and fibronectin binding by treatment with the cyclic RGD peptide and fibronectin knockdown induced tubular formation in the presence of EGF alone, but a fibronectin coat suppressed Wnt3a- and EGF-induced tubular formation. These results suggest that Wnt3a- and EGF-induced P2Y2R expression causes tubular formation by preventing the binding of integrins and fibronectin rather than by mediating nucleotide responses.
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Factor de Crecimiento Epidérmico/metabolismo , Células Epiteliales/metabolismo , Integrinas/metabolismo , Morfogénesis/fisiología , Receptores Purinérgicos P2Y2/metabolismo , Proteína Wnt3A/metabolismo , Animales , Línea Celular , Proliferación Celular/fisiología , Fibronectinas/metabolismo , Células HEK293 , Humanos , Nucleótidos/metabolismo , Ratas , Transducción de Señal/fisiologíaRESUMEN
Polycystic kidney diseases are characterized by numerous renal cysts that continuously enlarge resulting in compression of intact nephrons and tissue hypoxia. Recently, we have shown that hypoxia-inducible factor (HIF)-1α promotes secretion-dependent cyst expansion, presumably by transcriptional regulation of proteins that are involved in calcium-activated chloride secretion. Here, we report that HIF-1α directly activates expression of the purinergic receptor P2Y2R in human primary renal tubular cells. In addition, we found that P2Y2R is highly expressed in cyst-lining cells of human ADPKD kidneys as well as PKD1 orthologous mouse kidneys. Knockdown of P2Y2R in renal collecting duct cells inhibited calcium-dependent chloride secretion in Ussing chamber analyses. In line with these findings, knockdown of P2Y2R retarded cyst expansion in vitro and prevented ATP- and HIF-1α-dependent cyst growth. In conclusion, P2Y2R mediates ATP-dependent cyst growth and is transcriptionally regulated by HIF-1α. These findings provide further mechanistic evidence on how hypoxia promotes cyst growth.
Asunto(s)
Quistes/metabolismo , Células Epiteliales/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Túbulos Renales Proximales/metabolismo , Receptores Purinérgicos P2Y2/metabolismo , Animales , Quistes/patología , Células Epiteliales/citología , Femenino , Humanos , Túbulos Renales Proximales/citología , Masculino , Ratones , Ratones Noqueados , Persona de Mediana EdadRESUMEN
Esophageal cancer is a prominent worldwide illness that is divided into two main subtypes: esophageal squamous cell carcinoma and esophageal adenocarcinoma. Mortality rates are alarming, and the understanding of the mechanisms involved in esophageal cancer development, becomes essential. Purinergic signaling is related to many diseases and among these various types of tumors. Here we studied the effects of the P2Y2 receptor activation in different types of esophageal cancer. Esophageal tissue samples of healthy controls were used for P2Y2R expression quantification. Two human esophageal cancer cell lines Kyse-450 (squamous cell carcinoma) and OE-33 (adenocarcinoma) were used to perform in vitro analysis of cell proliferation, migration, adhesion, and the signaling pathways involved in P2Y2R activation. Data showed that P2Y2R was expressed in biopsies of patients with ESCC and adenocarcinoma, as well as in the two human esophageal cancer cell lines studied. The RT-qPCR analysis demonstrated that OE-33 cells have higher P2RY2 expression than Kyse-450 squamous cell line. Results showed that P2Y2R activation, induced by ATP or UTP, promoted esophageal cancer cells proliferation and colony formation. P2Y2R blockage with the selective antagonist, AR-C 118925XX, led to decreased proliferation, colony formation and adhesion. Treatments with ATP or UTP activated ERK 1/2 pathway in ESCC and ECA cells. The P2Y2R antagonism did not alter the migration of esophageal cancer cells. Interestingly, the esophageal cancer cell lines presented a distinct profile of nucleotide hydrolysis activity. The modulation of P2Y2 receptors may be a promising target for esophageal cancer treatment.
Asunto(s)
Adenocarcinoma/enzimología , Carcinoma de Células Escamosas/enzimología , Proliferación Celular/efectos de los fármacos , Neoplasias Esofágicas/enzimología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Agonistas del Receptor Purinérgico P2Y/farmacología , Receptores Purinérgicos P2Y2/efectos de los fármacos , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/patología , Adenosina Trifosfato/farmacología , Anciano , Anciano de 80 o más Años , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/patología , Adhesión Celular/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Neoplasias Esofágicas/tratamiento farmacológico , Neoplasias Esofágicas/patología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Fosforilación , Antagonistas del Receptor Purinérgico P2Y/farmacología , Receptores Purinérgicos P2Y2/metabolismo , Transducción de Señal , Uridina Trifosfato/farmacologíaRESUMEN
OBJECTIVE: Diabetic nephropathy (DN) is one of the most common complications of diabetes and a critical risk factor for developing end-stage renal disease. Activation of purinergic receptors, including P2Y2R has been associated with the pathogenesis of renal diseases, such as polycystic kidney and glomerulonephritis. However, the role of P2Y2R and its precise mechanisms in DN remain unknown. We hypothesised that P2Y2R deficiency may play a protective role in DN by modulating the autophagy signalling pathway. METHODS: We used a mouse model of DN by combining a treatment of high-fat diet and streptozotocin after unilateral nephrectomy in wild-type or P2Y2R knockout mice. We measured renal functional parameter in plasma, examined renal histology, and analysed expression of autophagy regulatory proteins. RESULTS: Hyperglycaemia and ATP release were induced in wild type-DN mice and positively correlated with renal dysfunction. Conversely, P2Y2R knockout markedly attenuates albuminuria, podocyte loss, development of glomerulopathy, renal tubular injury, apoptosis and interstitial fibrosis induced by DN. These protective effects were associated with inhibition of AKT-mediated FOXO3a (forkhead box O3a) phosphorylation and induction of FOXO3a-induced autophagy gene transcription. Furthermore, inhibitory phosphorylation of ULK-1 was decreased, and the downstream Beclin-1 autophagy signalling was activated in P2Y2R deficiency. Increased SIRT-1 (sirtuin-1) and FOXO3a expression in P2Y2R deficiency also enhanced autophagy response, thereby ameliorating renal dysfunction in DN. CONCLUSIONS: P2Y2R contributes to the pathogenesis of DN by impairing autophagy and serves as a therapeutic target for treating DN.
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Autofagia/fisiología , Nefropatías Diabéticas/metabolismo , Receptores Purinérgicos P2Y2/metabolismo , Animales , Apoptosis , Autofagia/genética , Diabetes Mellitus Experimental/metabolismo , Nefropatías Diabéticas/fisiopatología , Modelos Animales de Enfermedad , Proteína Forkhead Box O3/metabolismo , Riñón/metabolismo , Ratones , Ratones Noqueados , Podocitos/patología , Receptores Purinérgicos P2Y2/genética , Transducción de Señal , Estreptozocina/farmacologíaRESUMEN
Neurodegenerative diseases (ND) are a heterogeneous group of neurological disorders characterized by a progressive loss of neuronal function which results in neuronal death. Although a specific toxic factor has been identified for each ND, all of them share common pathological molecular mechanisms favouring the disease development. In the final stages of ND, patients become unable to take care of themselves and decline to a total functional incapacitation that leads to their death. Some of the main factors which contribute to the disease progression include proteasomal dysfunction, neuroinflammation, synaptic alterations, protein aggregation, and oxidative stress. Over recent years, evidence has been accumulated to suggest that purinergic signaling plays a key role in the aforementioned molecular pathways. In this review, we revise the implications of the purinergic signaling in the common molecular mechanism underlying the ND. In particular, we focus on the role of the purinergic receptors P2X7, P2Y2 and the ectoenzyme tissue-nonspecific alkaline phosphatase (TNAP).
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Enfermedades Neurodegenerativas/metabolismo , Nucleótidos/metabolismo , Fosfatasa Alcalina/metabolismo , Fosfatasa Alcalina/fisiología , Animales , Encéfalo/metabolismo , Humanos , Enfermedades Neurodegenerativas/patología , Enfermedades Neurodegenerativas/terapia , Neuronas/metabolismo , Nucleótidos/fisiología , Receptores Purinérgicos P2X7/metabolismo , Receptores Purinérgicos P2X7/fisiología , Receptores Purinérgicos P2Y2/metabolismo , Receptores Purinérgicos P2Y2/fisiología , Transducción de SeñalRESUMEN
Adenosine triphosphate (ATP) is stored as lysosomal vesicles in marginal cells of the stria vascular in neonatal rats, but the mechanisms of ATP release are unclear. Primary cultures of marginal cells from 1-day-old Sprague-Dawley rats were established. P2Y2 receptor and inositol 1,4,5-trisphosphate (IP3) receptor were immunolabelled in marginal cells of the stria vascular. We found that 30 µM ATP and 30 µM uridine triphosphate (UTP) evoked comparable significant increases in the intracellular Ca2+ concentration ([Ca2+]i) in the absence of extracellular Ca2+, whereas the response was suppressed by 100 µM suramin, 10 µM 1-(6-(17ß-3-methoxyester-1,3,5(10)-trien-17-yl)amino)-hexyl)-1H-pyrrole-2,5-dione(U-73122), 100 µM 2-aminoethoxydiphenyl borate (2-APB) and 5 µM thapsigargin (TG), thus indicating that ATP coupled with the P2Y2R-PLC-IP3 pathway to evoke Ca2+ release from the endoplasmic reticulum (ER). Incubation with 200 µM Gly-Phe-ß-naphthylamide (GPN) selectively disrupted lysosomes and caused significant increases in [Ca2+]I; this effect was partly inhibited by P2Y2R-PLC-IP3 pathway antagonists. After pre-treatment with 5 µM TG, [Ca2+]i was significantly lower than that after treatment with P2Y2R-PLC-IP3 pathway antagonists under the same conditions, thus indicating that lysosomal Ca2+ triggers Ca2+ release from ER Ca2+ stores. Baseline [Ca2+]i declined after treatment with the Ca2+ chelator 50 µM bis-(aminophenolxy) ethane-N,N,N',N'-tetra-acetic acid acetoxyme-thyl ester (BAPTA-AM) and 4 IU/ml apyrase. 30 µM ATP decrease of the number of quinacrine-positive vesicles via lysosome exocytosis, whereas the number of lysosomes did not change. However, lysosome exocytosis was significantly suppressed by pre-treatment with 5 µM vacuolin-1. Release of ATP and ß-hexosaminidase both increased after treatment with 200 µM GPN and 5 µM TG, but decreased after incubation with 50 µM BAPTA-AM, 4 IU/ml apyrase and 5 µM vacuolin-1. We suggest that ATP triggers Ca2+ release from the ER, thereby contributing to secretion of lysosomal ATP via lysosomal exocytosis. Lysosomal stored Ca2+ triggers Ca2+ release from the ER directly though the IP3 receptors, and lysosomal ATP evokes Ca2+ signals indirectly via the P2Y2R-PLC-IP3 pathway.
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Adenosina Trifosfato/metabolismo , Exocitosis , Lisosomas/metabolismo , Receptores Purinérgicos P2Y2/metabolismo , Adenosina Trifosfato/antagonistas & inhibidores , Animales , Animales Recién Nacidos , Calcio/metabolismo , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Exocitosis/efectos de los fármacos , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Lisosomas/efectos de los fármacos , Ratas , Ratas Sprague-DawleyRESUMEN
Sin Nombre virus (SNV) causes hantavirus cardiopulmonary pulmonary syndrome (HCPS) with the loss of pulmonary vascular endothelial integrity, and pulmonary edema without causing cytopathic effects on the vascular endothelium. HCPS is associated primarily with a dysregulated immune response. We previously found occult signs of hemostatic imbalance in the form of a sharp >30-100 fold increase in the expression of plasminogen activator inhibitor type 1 (PAI-1), in serial blood plasma draws of terminal stage-patients. However, the mechanism of the increase in PAI-1 remains unclear. PAI-1 is a primary inhibitor of fibrinolysis caused by tissue plasminogen activator (tPA) and urokinase plasminogen activator plasma (uPA). Here, we investigate factors that contribute to PAI-1 upregulation during HCPS. Using zymography, we found evidence of PAI-1-refractory uPA activity and no tPA activity in plasma samples drawn from HCPS patients. The sole prevalence of uPA activity suggested that severe inflammation drove PAI-1 activity. We have recently reported that the P2Y2 receptor (P2Y2R) mediates SNV infectivity by interacting in cis with ß3 integrins, which activates the latter during infection. P2Y2R is a known effector for several biological processes relevant to HCPS pathogenesis, such as upregulation of tissue factor (TF), a primary initiator of the coagulation cascade, stimulating vascular permeability and leukocyte homing to sites of infection. As P2Y2R is prone to upregulation under conditions of inflammation, we compared the expression level of P2Y2R in formalin fixed tissues of HCPS decedents using a TaqMan assay and immunohistochemistry. Our TaqMan results show that the expression of P2Y2R is upregulated significantly in HCPS cases compared to non- HCPS controls (P < 0.001). Immunohistochemistry showed that lung macrophages were the primary reservoir of high and coincident localization of P2Y2R, uPA, PAI-1, and TF antigens. We also observed increased staining for SNV antigens in the same tissue segments where P2Y2R expression was upregulated. Conversely, sections of low P2Y2R expression showed weak manifestations of macrophages, SNV, PAI-1, and TF. Coincident localization of P2Y2R and PAI-1 on macrophage deposits suggests an inflammation-dependent mechanism of increasing pro-coagulant activity in HCPS in the absence of tissue injury.
Asunto(s)
Infecciones por Hantavirus , Orthohantavirus/patogenicidad , Inhibidor 1 de Activador Plasminogénico/metabolismo , Receptores Purinérgicos P2Y2/metabolismo , Regulación hacia Arriba , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Adulto , Anciano , Permeabilidad Capilar , Femenino , Fibrinólisis , Infecciones por Hantavirus/diagnóstico por imagen , Infecciones por Hantavirus/inmunología , Infecciones por Hantavirus/patología , Síndrome Pulmonar por Hantavirus/diagnóstico por imagen , Síndrome Pulmonar por Hantavirus/inmunología , Síndrome Pulmonar por Hantavirus/patología , Humanos , Inmunohistoquímica , Inflamación , Leucocitos , Pulmón/diagnóstico por imagen , Pulmón/patología , Masculino , Persona de Mediana Edad , New Mexico , Transducción de Señal , Activador de Tejido Plasminógeno , Activador de Plasminógeno de Tipo Uroquinasa/sangreRESUMEN
BACKGROUND AND AIMS: The internalization of aggregated low-density lipoproteins (agLDL) mediated by low-density lipoprotein receptor related protein (LRP1) may involve the actin cytoskeleton in ways that differ from the endocytosis of soluble LDL by the LDL receptor (LDLR). This study aims to define novel mechanisms of agLDL uptake through modulation of the actin cytoskeleton, to identify molecular targets involved in foam cell formation in vascular smooth muscle cells (VSMCs). The critical observation that formed the basis for these studies is that under pathophysiological conditions, nucleotide release from blood-derived and vascular cells activates SMC P2Y2 receptors (P2Y2Rs) leading to rearrangement of the actin cytoskeleton and cell motility. Therefore, we tested the hypothesis that P2Y2R activation mediates agLDL uptake by VSMCs. METHODS: Primary VSMCs were isolated from aortas of wild type (WT) C57BL/6 and.P2Y2R-/- mice to investigate whether P2Y2R activation modulates LRP1 expression. Cells were transiently transfected with cDNA encoding a hemagglutinin-tagged (HA-tagged) WT P2Y2R, or a mutant P2Y2R that unlike the WT P2Y2R does not bind the cytoskeletal actin-binding protein filamin-A (FLN-A). RESULTS: P2Y2R activation significantly increased agLDL uptake, and LRP1 mRNA expression decreased in P2Y2R-/- VSMCs versus WT. SMCs, expressing P2Y2R defective in FLN-A binding, exhibit 3-fold lower LDLR expression levels than SMCs expressing WT P2Y2R, while cells transfected with WT P2Y2R show greater agLDL uptake in both WT and P2Y2R-/- VSMCs versus cells transfected with the mutant P2Y2R. CONCLUSIONS: Together, these results show that both LRP1 and LDLR expression and agLDL uptake are regulated by P2Y2R in VSMCs, and that agLDL uptake due to P2Y2R activation is dependent upon cytoskeletal reorganization mediated by P2Y2R binding to FLN-A.
Asunto(s)
Filaminas/metabolismo , Lipoproteínas LDL/sangre , Miocitos del Músculo Liso/metabolismo , Receptores de LDL/metabolismo , Receptores Purinérgicos P2Y2/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Actinas/metabolismo , Animales , Aorta/metabolismo , Movimiento Celular , Células Cultivadas , Citoesqueleto/metabolismo , Relación Dosis-Respuesta a Droga , Endocitosis , Células Espumosas/metabolismo , Humanos , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Microfilamentos/metabolismo , Músculo Liso Vascular/citología , Mutación , Transducción de Señal , Uridina Trifosfato/químicaRESUMEN
Lipoprotein oxidation, inflammation, and immune responses involving the vascular endothelium and immune cells contribute to the pathogenesis of atherosclerosis. In an atherosclerotic animal model, P2Y2 receptor (P2Y2R) upregulation and stimulation were previously shown to induce intimal hyperplasia and increased intimal monocyte infiltration. Thus, we investigated the role of P2Y2R in oxidized low-density lipoprotein (oxLDL)-mediated oxidative stress and the subsequent interaction between endothelial cells (ECs) and immune cells. The treatment of human ECs with oxLDL caused the rapid release of ATP (maximum after 5 min). ECs treated with oxLDL or the P2Y2R agonists ATP/UTP for 1h exhibited significant reactive oxygen species (ROS) production, but this effect was not observed in P2Y2R siRNA-transfected ECs. In addition, oxLDL and ATP/UTP both induced RAGE expression, which was P2Y2R dependent. Oxidized LDL- and ATP/UTP-mediated ROS production was diminished in RAGE siRNA-transfected ECs, suggesting that RAGE is an important mediator in P2Y2R-mediated ROS production. Treatment with oxLDL for 24h induced P2Y2R expression in the human monocyte cell line THP-1 and increased THP-1 cell migration toward ECs. The addition of apyrase, an enzyme that hydrolyzes nucleotides, or diphenyleneiodonium (DPI), a well-known inhibitor of NADPH oxidase, significantly inhibited the increase in cell migration caused by oxLDL. P2Y2R siRNA-transfected THP-1 cells did not migrate in response to oxLDL or ATP/UTP treatment, indicating a critical role for P2Y2R and nucleotide release in oxLDL-induced monocyte migration. Last, oxLDL and ATP/UTP effectively increased ICAM-1 and VCAM-1 expression and the subsequent binding of THP-1 cells to ECs, which was inhibited by pretreatment with DPI or by siRNA against P2Y2R or RAGE, suggesting that P2Y2R is an important mediator in oxLDL-mediated monocyte adhesion to ECs through the regulation of ROS-dependent adhesion molecule expression in ECs. Taken together, our findings suggest that P2Y2R could be a therapeutic target for the prevention of vascular disorders, including atherosclerosis.
Asunto(s)
Aterosclerosis/metabolismo , Células Endoteliales/metabolismo , Receptor para Productos Finales de Glicación Avanzada/biosíntesis , Receptores Purinérgicos P2Y/biosíntesis , Adenosina Trifosfato/administración & dosificación , Aterosclerosis/patología , Aterosclerosis/terapia , Línea Celular , Humanos , Molécula 1 de Adhesión Intercelular/biosíntesis , Lipoproteínas LDL/administración & dosificación , Lipoproteínas LDL/metabolismo , Terapia Molecular Dirigida , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Uridina Trifosfato/administración & dosificación , Molécula 1 de Adhesión Celular Vascular/biosíntesisRESUMEN
Sepsis is a severe systemic inflammatory response that is associated with high morbidity and mortality. A previous study using an animal model of sepsis showed that survival was significantly lower in WT mice than in P2Y(2) receptor (P2Y(2)R)-deficient mice, suggesting that P2Y(2)R plays a role in septic death. We therefore investigated the role of P2Y(2)R in the inflammatory responses of RAW264.7 murine macrophages to LPS. LPS time-dependently upregulated P2Y(2)R mRNA levels, with a prominent increase observed at 4 h. In addition, LPS increased ATP release in a time dependent manner (5-120 min post LPS treatment). Accordingly, we pretreated cells with LPS for 4 h to induce P2Y(2)R expression and then stimulated the cells with UTP or ATP for 16 h. Interestingly, ATP- or UTP-dependent P2Y(2)R activation in LPS-pretreated cells resulted in dramatically enhanced HMGB1 secretion, COX-2 and iNOS expression, and furthermore PGE2 and NO production compared to LPS treatment alone (4 h) or ATP or UTP treatment alone (16 h), an effect that was inhibited by P2Y(2)R silencing. In addition, these increases in HMGB1 secretion, COX-2 and iNOS expression and PGE(2) and NO production commonly involved the JNK, PKC and PDK pathways. Taken together, these data demonstrate that LPS-dependent upregulation of P2Y(2)R plays a critical role in facilitating the inflammatory responses induced by LPS.