title: The P2X7 receptor directly interacts with the NLRP3 inflammasome scaffold protein.
abstract: The P2X7 receptor (P2X7R) is a known and powerful activator of the NOD-like receptor (NLR)P3 inflammasome; however, the underlying pathways are poorly understood. Thus, we investigated the molecular mechanisms involved. The effect of P2X7R expression and activation on NLRP3 expression and recruitment was investigated by Western blot, RT-PCR, coimmunoprecipitation, and confocal microscopy in microglial mouse cell lines selected for reduced P2X7R expression and in primary cells from P2X7R(-/-) C57BL/6 mice. We show here that P2X7R activation by ATP (EC₅₀ = 1 mM) or benzoyl-ATP (EC₅₀ = 300 μM) and P2X7R down-modulation caused a 2- to 8-fold up-regulation of NLRP3 mRNA in mouse N13 microglial cells. Moreover, NLRP3 mRNA was also up-regulated in primary microglial and macrophage cells from P2X7R(-/-) mice. Confocal microscopy and immunoprecipitation assays showed that P2X7R and NLRP3 closely interacted at discrete subplasmalemmal sites. Finally, P2X7R stimulation caused a transient (3-4 min) cytoplasmic Ca(2+) increase localized to small (2-3 µm wide) discrete subplasmalemmal regions. The Ca(2+) increase drove P2X7R recruitment and a 4-fold increase in P2X7R/NLRP3 association within 1-2 min. These data show a close P2X7R and NLRP3 interaction and highlight the role of P2X7R in the localized cytoplasmic ion changes responsible for both NLRP3 recruitment and activation.
authors.affiliation: *Department of Morphology, Surgery and Experimental Medicine, Section of Pathology, Oncology and Experimental Biology, Department of Medical Sciences, Section of Internal Medicine, Gerontology, and Clinical Nutrition, and Laboratory of Technologies for Advanced Therapies, University of Ferrara, Ferrara, Italy.
authors.forename: Alessia
authors.initials: A
authors.lastname: Franceschini
authors.affiliation: *Department of Morphology, Surgery and Experimental Medicine, Section of Pathology, Oncology and Experimental Biology, Department of Medical Sciences, Section of Internal Medicine, Gerontology, and Clinical Nutrition, and Laboratory of Technologies for Advanced Therapies, University of Ferrara, Ferrara, Italy.
authors.forename: Marina
authors.initials: M
authors.lastname: Capece
authors.affiliation: *Department of Morphology, Surgery and Experimental Medicine, Section of Pathology, Oncology and Experimental Biology, Department of Medical Sciences, Section of Internal Medicine, Gerontology, and Clinical Nutrition, and Laboratory of Technologies for Advanced Therapies, University of Ferrara, Ferrara, Italy.
authors.forename: Paola
authors.initials: P
authors.lastname: Chiozzi
authors.affiliation: *Department of Morphology, Surgery and Experimental Medicine, Section of Pathology, Oncology and Experimental Biology, Department of Medical Sciences, Section of Internal Medicine, Gerontology, and Clinical Nutrition, and Laboratory of Technologies for Advanced Therapies, University of Ferrara, Ferrara, Italy.
authors.forename: Simonetta
authors.initials: S
authors.lastname: Falzoni
authors.affiliation: *Department of Morphology, Surgery and Experimental Medicine, Section of Pathology, Oncology and Experimental Biology, Department of Medical Sciences, Section of Internal Medicine, Gerontology, and Clinical Nutrition, and Laboratory of Technologies for Advanced Therapies, University of Ferrara, Ferrara, Italy.
authors.forename: Juana Maria
authors.initials: JM
authors.lastname: Sanz
authors.affiliation: *Department of Morphology, Surgery and Experimental Medicine, Section of Pathology, Oncology and Experimental Biology, Department of Medical Sciences, Section of Internal Medicine, Gerontology, and Clinical Nutrition, and Laboratory of Technologies for Advanced Therapies, University of Ferrara, Ferrara, Italy.
authors.forename: Alba Clara
authors.initials: AC
authors.lastname: Sarti
authors.affiliation: *Department of Morphology, Surgery and Experimental Medicine, Section of Pathology, Oncology and Experimental Biology, Department of Medical Sciences, Section of Internal Medicine, Gerontology, and Clinical Nutrition, and Laboratory of Technologies for Advanced Therapies, University of Ferrara, Ferrara, Italy.
authors.forename: Massimo
authors.initials: M
authors.lastname: Bonora
authors.affiliation: *Department of Morphology, Surgery and Experimental Medicine, Section of Pathology, Oncology and Experimental Biology, Department of Medical Sciences, Section of Internal Medicine, Gerontology, and Clinical Nutrition, and Laboratory of Technologies for Advanced Therapies, University of Ferrara, Ferrara, Italy.
authors.forename: Paolo
authors.initials: P
authors.lastname: Pinton
authors.affiliation: *Department of Morphology, Surgery and Experimental Medicine, Section of Pathology, Oncology and Experimental Biology, Department of Medical Sciences, Section of Internal Medicine, Gerontology, and Clinical Nutrition, and Laboratory of Technologies for Advanced Therapies, University of Ferrara, Ferrara, Italy fdv@unife.it.
authors.forename: Francesco
authors.initials: F
authors.lastname: Di Virgilio
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D002478:Cells, Cultured; D003593:Cytoplasm / Q000187:drug effects / Q000378:metabolism; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D006801:Humans; D058847:Inflammasomes / Q000378:metabolism*; D008264:Macrophages / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D017628:Microglia / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D018613:Microscopy, Confocal; D000071199:NLR Family, Pyrin Domain-Containing 3 Protein; D011485:Protein Binding; D058486:Receptors, Purinergic P2X7 / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015854:Up-Regulation / Q000187:drug effects
chemical_list: D002352:Carrier Proteins; D058847:Inflammasomes; D000071199:NLR Family, Pyrin Domain-Containing 3 Protein; C492716:Nlrp3 protein, mouse; D058486:Receptors, Purinergic P2X7; C033901:3'-O-(4-benzoyl)benzoyladenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D002118:Calcium
keywords: extracellular ATP; inflammation; purinergic receptors