P2X7 Receptor Protein Expression Research Articles

Functional expression of the ATP-gated P2X7 receptor in human iPSC-derived astrocytes

Activation of the ATP-gated P2X7 receptor (P2X7R), implicated in numerous diseases of the brain, can trigger diverse responses such as the release of pro-inflammatory cytokines, modulation of neurotransmission, cell proliferation or cell death. However, despite the known species-specific differences in its pharmacological properties, to date, most functional studies on P2X7R responses have been analyzed in cells from rodents or immortalised cell lines. To assess the endogenous and functional expression of P2X7Rs in human astrocytes, we differentiated human-induced pluripotent stem cells (hiPSCs) into GFAP and S100 β-expressing astrocytes. Immunostaining revealed prominent punctate P2X7R staining. P2X7R protein expression was also confirmed by Western blot. Importantly, stimulation with the potent non-selective P2X7R agonist 2′,3′-O-(benzoyl-4-benzoyl)-adenosine 5′- triphosphate (BzATP) or endogenous agonist ATP induced robust calcium rises in hiPSC-derived astrocytes which were blocked by the selective P2X7R antagonists AFC-5128 or JNJ-47965567. Our findings provide evidence for the functional expression of P2X7Rs in hiPSC-derived astrocytes and support their in vitro utility in investigating the role of the P2X7R and drug screening in disorders of the central nervous system (CNS).

Blockage of the P2X7 Receptor Attenuates Harmful Changes Produced by Ischemia and Reperfusion in the Myenteric Plexus.

Our work analyzed the effects of a P2X7 receptor antagonist, Brilliant Blue G (BBG), on rat ileum myenteric plexus following ischemia and reperfusion (ISR) induced by 45min of ileal artery occlusion with an atraumatic vascular clamp with 24h (ISR 24-h group) or 14 d of reperfusion (ISR 14-d group). Either BBG (50mg/kg or 100mg/kg, BBG50 or BBG100 groups) or saline (vehicle) was administered subcutaneously 1h after ischemia in the ISR 24-h group or once daily for the 5 d after ischemia in the ISR 14-d group (n = 5 per group). We evaluated the neuronal density and profile area by examining the number of neutrophils in the intestinal layers, protein expression levels of the P2X7 receptor, intestinal motility and immunoreactivity for the P2X7 receptor, nitric oxide synthase, neurofilament-200, and choline acetyl transferase in myenteric neurons. The neuronal density and profile area were restored by BBG following ISR. The ischemic groups showed alterations in P2X7 receptor protein expression and the number of neutrophils in the intestine and decreased intestinal motility, all of which were recovered by BBG treatment. We concluded that ISR morphologically and functionally affected the intestine and that its effects were reversed by BBG treatment, suggesting the P2X7 receptor as a therapeutic target.

Inhibition of NLRP3 inflammasome: a new protective mechanism of cinnamaldehyde in endotoxin poisoning of mice

Context: Cinnamaldehyde (CA) has a protective effect in endotoxin poisoning of mice, but there is no direct evidence for the protective effect of CA through inhibition of NLRP3 inflammasome activation in endotoxin poisoning of mice.Objective: We aimed to investigate the protective mechanism of CA in endotoxin poisoned mice through NLRP3 inflammasome.Materials and methods: First, we evaluated the anti-inflammatory effect of CA in phorbol-12-myristate acetate–differentiated THP-1 cells through the NLRP3 inflammasome. Second, in a mouse model of lipopolysaccharide (LPS)-induced endotoxin poisoning, CA was administrated for 5 d (once a day) before the 15 mg/kg LPS challenge. Then, the levels of IL-1β in serum were measured, and the effect of CA on the NLRP3 inflammasome activation and the expression of cathepsin B and P2X7R proteins in lung were explored.Results: In vitro, CA decreased the levels of p20, pro-IL-1β and IL-1β in cell culture supernatants, as well as the expression of NLRP3 and IL-1β mRNA in cells. In vivo, CA decreased IL-1β production in serum. Furthermore, CA suppressed LPS-induced NLRP3, p20, Pro-IL-1β, P2X7 receptor (P2X7R) and cathepsin B protein expression in lung, as well as the expression of NLRP3 and IL-1β mRNA.Conclusions: CA has a protective effect in the endotoxin poisoned mice through the inhibition of NLRP3 inflammasome activation. Furthermore, CA suppresses the NLRP3 inflammasome activation by inhibiting the expression of cathepsin B and P2X7R protein expression. CA can be considered as a potential therapeutic candidate for diseases involved in endotoxin poisoning such as sepsis.

Effect of NaHS on ATP-induced P2X receptor expression in rat microglia

To observed the effect of sodium hydrosulphide (NaHS), a donor of H2S on the cell viability,the membrane permeability and the expression of P2X7 receptor induced by adenosine triphosphate(ATP) in rat microglia. Rat microglia in logarithmic growth phase was randomly divided into 4 groups. In control group, the cells were cultured without ATP treatment. In ATP group, the cells were treatment with ATP after cultured for 24 hours. In NaHS+ATP group, the cells were incubated with NaHS for 30 min before ATP, and NaHS always existed in the reaction system. In KN-62+ATP group, the cells were pretreated with KN-62 for 30 min, the others were as the same as NaHS+ATP group. The cell viability was detected by MTT. Fluorescent dyes YO-PRO-1 was used to observe the membrane permeability. The expression of P2X7 receptor was examined by immunofluorescence staining. ① Compared with control group, the cell viability dropped after treatment with ATP (1、3、5、10 mmol/L) for 3 hours. When pre-incubation with NaHS(200 μmol/L), the cell viability was apparently higher than that of ATP alone group(P<0.01), while 400 μmol/L had no further beneficial.②The YO-PRO-1 fluorescence intensity was obviously elevated by ATP in rat microglia, but this effect was counteracted by NaHS pretreatment (P<0.01). ③ The expression of P2X7 receptor protein was significantly increased after ATP(3 mmol/L) for 3 h. While the expression upregulation of P2X7 receptor protein induced by ATP was significantly counteracted by pretreating with NaHS(200 μmol/L) (P<0.01). NaHS could reduce the expression of P2X7 receptor, decrease membrane permeability, and increase the cell viability in rat microglia injured by ATP. So the cytoprotection of hydrogen sulfide may be related to the expression and function of P2X7 receptor.

Activated Müller Cells Involved in ATP-Induced Upregulation of P2X7 Receptor Expression and Retinal Ganglion Cell Death.

P2X7 receptor (P2X7R), an ATP-gated ion channel, plays an important role in glaucomatous retinal ganglion cell (RGC) apoptotic death, in which activated retinal Müller glial cells may be involved by releasing ATP. In the present study, we investigated whether and how activated Müller cells may induce changes in P2X7R expression in RGCs by using immunohistochemistry and Western blot techniques. Intravitreal injection of DHPG, a group I metabotropic glutamate receptor (mGluR I) agonist, induced upregulation of GFAP expression, suggestive of Müller cell activation (gliosis), as we previously reported. Accompanying Müller cell activation, P2X7R protein expression was upregulated, especially in the cells of ganglion cell layer (GCL), which was reversed by coinjection of brilliant blue G (BBG), a P2X7R blocker. In addition, intravitreal injection of ATP also induced upregulation of P2X7R protein expression. Similar results were observed in cultured retinal neurons by ATP treatment. Moreover, both DHPG and ATP intravitreal injection induced a reduction in the number of fluorogold retrogradely labeled RGCs, and the DHPG effect was partially rescued by coinjection of BBG. All these results suggest that activated Müller cells may release ATP and, in turn, induce upregulation of P2X7R expression in the cells of GCL, thus contributing to RGC death.

The P2X7 receptor regulates cell survival, migration and invasion of pancreatic ductal adenocarcinoma cells.

BackgroundPancreatic ductal adenocarcinoma (PDAC) is presently one of the cancers with the worst survival rates and least effective treatments. Moreover, total deaths due to PDAC are predicted to increase in the next 15 years. Therefore, novel insights into basic mechanism of PDAC development and therapies are needed. PDAC is characterized by a complex microenvironment, in which cancer and stromal cells release different molecules, such as ATP. ATP can be transported and/or exocytosed from active cancer cells and released from dying cells in the necrotic core of the cancer. We hypothesized that one of the ATP receptors, the P2X7 receptor (P2X7R) could be an important player in PDAC behaviour.MethodsWe determined the expression (real time PCR and Western blot) and localization (immunofluorescence) of P2X7R in human PDAC cell lines (AsPC-1, BxPC-3, Capan-1, MiaPaCa-2, Panc-1) and a “normal” human pancreatic duct epithelial cell line (HPDE). The function of P2X7R in proliferation (BrdU assay), migration (wound assay) and invasion (Boyden chamber with matrigel) was characterized. Furthermore, we studied P2X7R-dependent pore formation (YoPro-1 assay) and cell death (caspase and annexin V / propidium iodide assays).ResultsWe found higher expression of P2X7R protein in PDAC compared to HPDE cells. P2X7R had notable disparate effects on PDAC survival. Firstly, high concentrations of ATP or the specific P2X7R agonist, BzATP, had cytotoxic effects in all cell lines, and cell death was mediated by necrosis. Moreover, the P2X7R–pore antagonist, A438079, prevented ATP-induced pore formation and cell death. Second, in basal conditions and with low concentrations of ATP/BzATP, the P2X7R allosteric inhibitor AZ10606120 reduced proliferation in all PDAC cell lines. P2X7R also affected other key characteristics of cancer cell behavior. AZ10606120 reduced cell migration and invasion in PDAC cell lines compared to that of untreated/vehicle-treated control cells, and stimulation with sub-millimolar concentrations of ATP or BzATP substantially increased cell invasion.ConclusionsPDAC cell lines overexpress P2X7R and the receptor plays crucial roles in cell survival, migration and invasion. Therefore, we propose that drugs targeting P2X7R could be exploited in therapy of pancreatic cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0472-4) contains supplementary material, which is available to authorized users.

P2X7 receptors are a potential novel target for anti-glioma therapies

Human gliomas pose significant morbidity and mortality to those afflicted by them, and currently there are no curative treatment modalities available for these highly invasive tumours. With the approval from the human ethics committee, patients diagnosed with brain tumour (glioma) were recruited for this study. At the time of surgical resection, freshly resected tumour as well as ‘peri-tumour’ tissue were taken directly from theatre to the laboratory and were successfully cultured. Confocal fluorescence microscopy techniques and immunohistochemistry were used for characterization of human glioma cultures. Dye uptake experiments and confocal microscopy were utilized for P2X7 receptor (P2X7R) pore activity. We reveal human glioma cultures to contain microglia in close association with glioma (tumour) cells. Both glioma cells and microglia were found to express the purinergic, ATP sensing, P2X7R. P2X7R protein expression was increased in microglia derived from tumour when compared to ‘peri-tumour’ tissue. The pore capacity of P2X7R in tumour-associated microglia was functional, as evidenced by dye uptake experiments. Importantly, inhibition of P2X7R with the synthetic antagonist, brilliant blue G (BBG) resulted in a significant decrease in the number of glioma cells in culture. P2X7R was found to be over-expressed in grade IV human gliomas and its pore capacity was functional. Antagonism of P2X7R with BBG resulted in a decrease in tumour cell number. This identifies P2X7R as a promising therapeutic target to combat human glioma proliferation.

Isoflurane-induced neuronal apoptosis in developing hippocampal neurons.

We hypothesized that the P2X7 receptor may be the target of isoflurane, so we investigated the roles of the P2X7 receptor and inositol triphosphate receptor in calcium overload and neuronal apoptosis induced by isoflurane in cultured embryonic rat hippocampal neurons. Results showed that isoflurane induced widespread neuronal apoptosis and significantly increased cytoplasmic Ca2+. Blockade of P2X7 receptors or removal of extracellular Ca2+ combined with blockade of inositol triphosphate receptors completely inhibited apoptosis or increase in cytoplasmic Ca2+. Removal of extracellular Ca2+ or blockade of inositol triphosphate receptor alone could partly inhibit these effects of isoflurane. Isoflurane could directly activate P2X7-gated channels and induce inward currents, but did not affect the expression of P2X7 receptor protein in neurons. These findings indicate that the mechanism by which isoflurane induced neuronal apoptosis in rat developing brain was mediated by intracellular calcium overload, which was caused by P2X7 receptor mediated calcium influx and inositol triphosphate receptor mediated calcium release.

Effects of RNA interference targeting P2X7 receptor on microglial phagocytosis of amyloid-β protein

To investigate the effect of silencing P2X7 receptor (P2X7R) by RNA interference on microglial phagocytosis of amyloid-β (Aβ) protein and to explore its possible mechanism. The small interfering RNA (siRNA) targeting the P2X7R gene was identified. The microglial cells activated by Aβ1-42 were infected with the Lipofectamine-siP2X7R and it was designated as siP2X7R group. Microglia infected with Lipofectamine-siNC was designated as siNC group and non-infected cells as con group. The levels of P2X7R mRNA were detected by real-time PCR and the P2X7R protein was determined by Western blotting. The levels of IL-1β and TNF-α were measured by ELISA. The microglial phagocytosis of Aβ1-42 was observed by ELISA and immunocytochemistry staining. Detected by the Real-time PCR, the expression level of P2X7R mRNA of siP2X7R group decreased significantly versus siNC and con groups (P<0.05). The lowered expression of P2X7R protein detected by Western blotting was consistent with Real-time PCR. After RNA interference silencing P2X7R, the levels of IL-1β and TNF-α detected by ELISA in siP2X7R group less than those in con, siNC groups, significantly (P<0.05). In con, siNC and siP2X7R groups respectively, the levels of Aβ1-42 in supernatant were (423±20) pg/ml, (417±16) pg/ml, (296±30) pg/ml and the levels of Aβ1-42 in the microglia were (190±37) pg/ml, (187±39) pg/ml, (322±26) pg/ml. Compared to siNC and con groups, in siP2X7R group the levels of Aβ1-42 in supernatant decreased (P<0.05) and the levels of Aβ1-42 in the microglia increased (P<0.05). Aβ1-42 immunofluorescence staining showed that the red fluorescent products were seen in the cytoplasm of most microglias in siP2X7R group, but in con or siNC groups in only few microglias these products were depicted. The silence expression of P2X7R by RNA interference effectively decreases the levels of IL-1β and TNF-α released by microglia and promotes microglia to phagocytose Aβ. P2X7R could be used as an effective therapeutic target for RNA interference treatment of Alzheimer's disease.

Regulation of lung surfactant secretion by microRNA-150

P2X7 receptor (P2X7R) is a purinergic ion-channel receptor. We have previously shown that the activation of P2X7R in alveolar type I cells stimulates surfactant secretion in alveolar type II cells. In this study, we determined whether miR-150 regulates P2X7R-mediated surfactant secretion. The miR-150 expression level in alveolar type II cells was much higher than alveolar type I cells, which was inversely correlated with the P2X7R protein level. An adenovirus expressing miR-150 significantly reduced the P2X7R protein expression in E10 cells, an alveolar type I cell line. Furthermore, pre-treatment of E10 cells with the adenovirus reduced the surfactant secretion induced by E10 cell conditioned medium. Our study demonstrates that miR-150 regulates surfactant secretion through P2X7R.

MICRORNA‐150 REGULATES SURFACTANT SECRETION VIA P2X7 RECEPTORS

P2X7 receptor (P2X7R) is a purinergic ion‐channel receptor. Previously, we found that the activation of P2X7R in alveolar type I cells could activate the surfactant secretion in alveolar epithelial type II cells. In this study, we determined whether miRNAs regulate P2X7R‐mediated surfactant secretion. We first used a dual‐luciferase assay to screen the regulation of P2X7R 3′‐UTR activity by miR‐150, miR‐186, miR‐204 and miR‐211. We found that miR‐150 is a regulator of P2X7R in lung epithelial cells. The miR‐150 regulation could be disrupted by site mutation of mature miR‐150 or mutation of P2X7R 3′‐UTR at miR‐150 binding sites. MiR‐150 expression level in alveolar type II cells was much higher than alveolar type I cells, which was inversely correlated with the P2X7R protein level. Adenovirus expressing miR‐150 significantly downregulated the P2X7R protein expression in E10 cells. Furthermore, pre‐treatment of E10 cells with adenovirus expressing miR‐150 disrupted the surfactant secretion stimulated by E10 cell conditioned media. Our study is the first of its kind to demonstrate that miR‐150 regulates P2X7R mediated surfactant secretion (supported by NIH grants R01 HL‐083188).