P2受体依赖的钙信号通路在骨保护素致破骨细胞黏附结构损伤中的作用机理

Purinergic signaling pathway mediated by P2 receptors is the upstream regulator of calcium signaling pathway. In the previous study, we found that Osteoprotegerin (OPG) disrupted the adhesion structure of osteoclasts via calcium signaling pathway, whereas activating P2 purinergic receptors with ATP could reverse the effects of OPG on osteoclast adhesion structure. The results indicated that P2 receptors might be the upstream target for OPG to induce the disruption of osteoclast adhesion structure. However, the mechanism of purinergic signaling in the regulation of calcium signaling pathway participating in OPG-induced disruption of osteoclast adhesion structure has been understood poorly. The mature osteoclasts are differentiated from bone marrow-derived macrophages in the duck embryo and Balb/c mouse treated with M-CSF and RANKL. After OPG treatment, the expression of P2 receptor isoforms is detected by RT-PCR and Western blotting to identify which P2 receptor isoform hosts response to OPG treatment. The adenovirus vector mediated transfection of P2 receptor genes into osteoclasts or broad-spectrum P2 receptor agonists/inhibitors are applied, in combination with intracellular Ca2+ chelating agent or CaM inhibitor and then OPG was added into the culture system. Flow cytometry, scanning electron microscope, Western blotting and laser scanning confocal microscopy is used to detect key molecules involving in calcium signaling pathway ([Ca2+]i, Ca2+ distribution, Ca2+ oscillation, Ca2+-ATPase activity and CaMK/CREB/NFATc1 signaling pathway related proteins) and adhesion structure (Lamellipodia, filopodia, podosome realated proteins including Pyk2, c-Src, Paxillin, p130cas, Cbl, and actin linked molecules like WASP, Cortactin and Arp2/3). The results will reveal the role of purinergic signaling pathway mediated by P2 receptors acted in the regulation of OPG-induced osteoclast adhesion structure disruption via calcium signaling pathway and clarify which P2 receptor is the upstream target for OPG to regulate osteoclast adhesion structure. Our research findings will help to improve the clinical application of OPG in curing bone metabolism and diseases of livestock and poultry caused by osteoclast overpopulation or excessive activation.

P2受体介导的嘌呤信号是Ca2+信号通路的上游调节器。本课题组前期研究表明骨保护素（OPG）可经Ca2+信号损伤破骨细胞黏附结构，而激活P2受体能抑制该作用，提示P2受体可能是OPG调控破骨细胞黏附结构的上游靶点。但P2受体介导的嘌呤信号在OPG致破骨细胞黏附结构损伤Ca2+信号通路中的调控机制尚未阐明。本项目以RANKL/M-CSF 诱导高邮鸭胚和Balb/c小鼠骨髓源巨噬细胞分化形成的破骨细胞为研究对象，OPG处理，检测P2受体，筛选出对OPG产生应答的受体；应用腺病毒转染过表达/沉默P2受体基因或P2受体广谱激动/抑制剂，结合Ca2+信号特异性抑制剂，OPG处理，检测Ca2+信号通路和黏附结构关键分子，揭示P2受体依赖的Ca2+信号通路在OPG致破骨细胞黏附结构损伤中的作用机理，明确哪些P2受体是OPG调控破骨细胞黏附的上游靶点，为临床应用OPG治疗畜禽骨代谢疾病提供依据。

P2X7受体参与调控破骨细胞（OCs）的黏附、融合及分化过程，是Ca2+信号通路的上游调节器。骨保护素（OPG）可经Ca2+信号损伤OCs黏附结构，抑制OCs分化，而激活P2受体能抑制该作用。本研究旨在阐明P2X7依赖的Ca2+信号通路在OPG 致OCs黏附结构损伤中的作用机理。重要的结果及意义如下：. Balb/c小鼠骨髓源巨噬细胞分化形成的OCs：（1）OPG显著抑制PYK2和SRC的磷酸化水平，并损伤OCs黏附结构，导致胞内Ca2+浓度下降，抑制CAM/CAMK磷酸化水平、PCG1β、CREB、PLC、c-Fos和NFATc1活性，降低PYK2和SRC之间的蛋白相互作用。BAPTA-AM和W-7预处理能有效地恢复SRC和PYK2的磷酸化水平及其结合力。表明OPG通过Ca2+信号调控OCs黏附。（2）OPG显著降低P2X7蛋白表达水平，促进ATP向胞外释放，抑制Ca2+-ATPase活性。敲减P2X7进一步加剧OPG对OCs黏附结构的损伤、分化的抑制和Ca2+信号通路的阻滞。而P2X7R激动剂BzATP发挥了相反的作用。表明OPG通过P2X7介导的Ca2+信号通路调控OCs黏附和分化。（3）OPG显著抑制MAPK家族ERK、JNK、p38磷酸化水平。Erk、JNK和P38抑制剂预处理显著缓解OPG导致的P2X7蛋白表达量的下降，抑制OPG对细胞黏附结构的破坏作用。BzATP与OPG共处理能够显著提高ERK、JNK和p-38的磷酸化水平，而P2X7RshRNA作用相反。表明P2X7在MAPK信号通路中起着正调控作用，OPG通过P2X7/MAPK信号通路损伤OCs黏附结构。. 高邮鸭胚骨髓源巨噬细胞分化形成的OCs：结果与鼠源OCs一致。