OBJECTIVETo clone the entire coding sequence and analyze the function of P2X7 receptor of J6-1 human leukemia cells.

METHODSThe entire coding sequence of P2X7 receptor was amplified by RT-PCR and then inserted into pTARGET plasmid to construct an eukaryotic expressing plasmid followed by DNA sequencing. HEK293 cells stably expressing P2X7 receptor were obtained after transfection and screening, and confirmed by RT-PCR and Western blotting. The bleb formation upon agonist stimulation was observed under phase contrast microscope.

RESULTSThe entire coding sequence of P2X7 receptor of J6-1 cells was successfully cloned. DNA sequencing analysis revealed a substitution of G559, for A559, causing a substitution of Glu187 for Gln187. The P2X7 receptor derived from J6-1 cells could be functionally expressed in HEK293 cells, in which bleb formation could be detected upon stimulation.

CONCLUSIONSThe entire coding sequence of P2X7 receptors was successfully cloned from J6-1 leukemia cells. Other unknown mechanism may contribute to the dysfunction of P2X7 receptor in these cells.

Cell Line, Tumor ; Cloning, Molecular ; DNA, Complementary ; genetics ; Gene Expression ; Humans ; Leukemia ; genetics ; metabolism ; Receptors, Purinergic P2 ; genetics ; physiology ; Receptors, Purinergic P2X7 ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection

Regulation of P2X7 receptor expression is of interest because activation of this receptor by extracellular ATP triggers a wide variety of cell functions in leukocytes. However, its expression and modulation in human peripheral blood mononuclear cells (PBMC) and monocytes remain unclear. RT-PCR was used to detect the constitutive level of P2X7 receptor and the levels upon stimulation with bacteria, bacterial product, mitogen and various cytokines in human PBMC and monocytes. P2X7 receptor mRNA was detected in PBMC and monocytes. P2X7 receptor expression in PBMC was up-regulated by interleukin-2, -4, -6 (IL-2, IL-4, IL-6) tumour necrosis factor-alpha (TNF-alpha), lipopolysaccharide (LPS) and heat-inactivated Staphylococcus aureus Cowan strain I (SAC). However, interferon-gamma (IFN-gamma), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF) and phytohemagglutinin-M (PHA-M) had little effect on the expression of P2X7 receptor. Furthermore, LPS and M-CSF could up-regulate P2X7 receptor expression in monocytes, while IFN-gamma, TNF-alpha and GM-CSF had weak effects, but pretreatment with these inducers could not further enhance LPS-stimulated P2X7 receptor expression in monocytes. The results obtained demonstrate that inflammatory stimuli drive P2X7 expression, thus supporting the hypothesis that P2X7 receptor may play a role in the inflammatory responses against bacteria infection, which need further verification.
Humans ; Interleukin-2 ; physiology ; Interleukin-4 ; physiology ; Leukocytes, Mononuclear ; drug effects ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Receptors, Purinergic P2X7 ; genetics ; metabolism ; Tumor Necrosis Factor-alpha ; physiology

Background: The nucleotide-binding and oligomerization domain-like receptor protein 3 (NLRP3) inflammasome composed of NLRP3, apoptosis-associated speck-like protein containing CARD (ASC), and caspase-1 is engaged in the inflammatory response of many kidney diseases and can be activated by purinergic 2X7 receptor (P2X7R). This study was conducted to explore whether P2X7R plays a pathogenic role in the podocyte damage of obesity-related glomerulopathy (ORG) and whether this role is mediated by the activation of NLRP3 inflammasome. Methods: A mouse model of ORG was established by high-fat diet feeding. The conditionally immortalized mouse podocytes were cultured with leptin or with leptin and P2X7R antagonist (KN-62 or A438079). The mRNA and protein expression of the P2X7R and NLRP3 inflammasome components including NLRP3, ASC, and caspase-1, as well as the podocyte-associated molecules including nephrin, podocin, and desmin in mouse renal cortex or cultured mouse podocytes were tested by real-time-polymerase chain reaction and Western blot analysis, respectively. Results: The significantly upregulated expression of P2X7R and NLRP3 inflammasome components and the NLRP3 inflammasome activation were observed in the renal cortex (in fact their location in podocytes was proved by confocal microscopy) of ORG mice in vivo, which were accompanied with the morphological changes of podocyte damage and the expression changes of podocyte-associated molecules. Similar changes in the expression of P2X7R and NLRP3 inflammasome components as well as in the expression of podocyte-associated molecules were also observed in the cultured podocyte studies treated by leptin in vitro, and all of the above changes were significantly attenuated by the P2X7R antagonist KN-62 or A438079. Conclusions P2X7R could trigger the activation of NLRP3 inflammasome, and the activated P2X7R/NLRP3 inflammasome in podocytes might be involved in the podocyte damage of ORG.
Animals ; Blotting, Western ; Body Weight ; physiology ; Inflammasomes ; metabolism ; Kidney Glomerulus ; metabolism ; pathology ; Male ; Mice ; Mice, Inbred C57BL ; NLR Family, Pyrin Domain-Containing 3 Protein ; genetics ; metabolism ; Obesity ; complications ; Podocytes ; metabolism ; pathology ; Receptors, Purinergic P2X7 ; genetics ; metabolism

P2X7 is the most important subtype of the ATP receptors known so far. Recent investigations showed that the downstream signaling pathway of P2X7 is coupled with several key inflammatory molecules including IL-1beta and IL-18, this suggests P2X7 might have roles in the inflammatory diseases. Moreover, attenuation of P2X7 by selective antagonists in vitro and knockout mice in vivo reducing the inflammatory response indicated that P2X7 is a potential therapeutic target for inflammatory diseases. However, most previous studies on P2X7 were focused on nerve system diseases most, while its effects in inflammatory respiratory diseases, especially in asthma, chronic obstructive pulmonary disease (COPD) and lung cancer have been poorly investigated. In this paper, we reviewed the research progress on the structure, distribution, biological activities of P2X7 and its relationship with inflammatory respiratory diseases including asthma, COPD and lung cancer, along with the development of P2X7 antagonist as therapeutics.
Animals ; Asthma ; drug therapy ; metabolism ; Humans ; Inflammation ; drug therapy ; metabolism ; Interleukin-18 ; metabolism ; Interleukin-1beta ; metabolism ; Lung Neoplasms ; drug therapy ; metabolism ; Mice ; Polymorphism, Single Nucleotide ; Pulmonary Disease, Chronic Obstructive ; drug therapy ; metabolism ; Purinergic P2X Receptor Antagonists ; therapeutic use ; Receptors, Purinergic P2X7 ; chemistry ; genetics ; metabolism ; Respiratory Tract Diseases ; drug therapy ; metabolism

The possibility that P2X7 receptor (P2X7R) expression in microglia would mediate neuronal damage via reactive oxygen species (ROS) production was examined in the APPswe/PS1dE9 mouse model of Alzheimer's disease (AD). P2X7R was predominantly expressed in CD11b-immunopositive microglia from 3 months of age before Abeta plaque formation. In addition, gp91phox, a catalytic subunit of NADPH oxidase, and ethidium fluorescence were detected in P2X7R-positive microglial cells of animals at 6 months of age, indicating that P2X7R-positive microglia could produce ROS. Postsynaptic density 95-positive dendrites showed significant damage in regions positive for P2X7R in the cerebral cortex of 6 month-old mice. Taken together, up-regulation of P2X7R activation and ROS production in microglia are parallel with Abeta increase and correlate with synaptotoxicity in AD.
Aging ; *Alzheimer Disease/genetics/metabolism/pathology ; Amyloid beta-Peptides ; Animals ; Antigens, CD11b/immunology ; Blotting, Western ; Cerebral Cortex/metabolism/*pathology ; Disease Models, Animal ; Gene Expression ; Mice ; Mice, Transgenic ; Microglia/*metabolism/pathology ; Neurons/metabolism/*pathology ; Plaque, Amyloid ; Reactive Oxygen Species/*metabolism ; Receptors, Immunologic/analysis ; Receptors, Purinergic P2X7/*genetics/metabolism