Franceschini Alessia, Capece Marina, Chiozzi Paola, Falzoni Simonetta, Sanz Juana Maria, Sarti Alba Clara, Bonora Massimo, Pinton Paolo, Di Virgilio Francesco
*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.
*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
FASEB J. 2015 Jun;29(6):2450-61. doi: 10.1096/fj.14-268714. Epub 2015 Feb 17.
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.
P2X7受体（P2X7R）是核苷酸结合寡聚化结构域样受体（NLR）P3炎性小体已知的强效激活剂；然而，其潜在机制尚不清楚。因此，我们对其中涉及的分子机制进行了研究。通过蛋白质免疫印迹法、逆转录-聚合酶链反应、免疫共沉淀以及共聚焦显微镜技术，在选取的P2X7R表达降低的小鼠小胶质细胞系以及来自P2X7R基因敲除（P2X7R(-/-)）C57BL/6小鼠的原代细胞中，研究了P2X7R表达及激活对NLRP3表达和募集的影响。我们在此表明，ATP（半数有效浓度[EC₅₀]=1 mM）或苯甲酰-ATP（EC₅₀=300 μM）激活P2X7R以及P2X7R下调，会使小鼠N13小胶质细胞中NLRP3 mRNA上调2至8倍。此外，来自P2X7R(-/-)小鼠的原代小胶质细胞和巨噬细胞中NLRP3 mRNA也上调。共聚焦显微镜和免疫沉淀分析表明，P2X7R与NLRP3在离散的质膜下位点紧密相互作用。最后，P2X7R刺激导致细胞质中钙离子（Ca(2+)）短暂（3至4分钟）增加，且局限于小的（宽2至3微米）离散质膜下区域。钙离子增加促使P2X7R募集，并在1至2分钟内使P2X7R/NLRP3结合增加4倍。这些数据表明P2X7R与NLRP3存在紧密相互作用，并突出了P2X7R在导致NLRP3募集和激活的局部细胞质离子变化中的作用。
