Department of Periodontology, Guanghua School of Stomatology, Hospital of Stomatology, Sun Yat-sen University, 56 Lingyuan Road West, Guangzhou, 510055, People's Republic of China.
Guangdong Provincial Key Laboratory of Stomatology, Guangzhou, China.
J Mol Histol. 2020 Apr;51(2):147-159. doi: 10.1007/s10735-020-09865-w. Epub 2020 Mar 19.
As a crucial virulence factor of Porphyromonas gingivalis, gingipains play an important role in periodontal destruction. This study aimed to investigate the effect of gingipains on osteoclastogenesis. We used RAW264.7 cells as osteoclast precursors in our study. In experimental groups, cells were treated with gingipains and/or receptor activator of nuclear factor-κB ligand (RANKL). Tartrate-resistant acid phosphatase (TRAP) activity staining assay showed osteoclast precursors and RANKL-induced mature osteoclasts were increased in a gingipains dose-dependent manner. Real-time reverse transcription polymerase chain reaction analysis demonstrated that gingipains upregulated osteoclastic genes including the protease cathepsin K (Ctsk), matrix metalloprotein 9 (Mmp9), nuclear factor of activated T cells 1 (Nfatc1) and acid phosphatase 5, tartrate resistant (Acp5) in a time-dependent manner. Western blotting assays presented upregulated expressions of TNF receptor-activating factor 6 (TRAF6) and integrin β3 induced by gingipains and RANKL compared to RANKL alone. Enhanced integrin-related signaling was also demonstrated by elevated phosphorylations of FAK and paxillin compared to control. Moreover, the pit resorption assays showed that gingipains augmented bone resorptive function of osteoclasts induced by RANKL. When we used Cilengitide to block integrin αvβ3, gingipains reversed the reduction of formation and resorptive function in RANKL-induced osteoclasts, as they enhanced integrin αvβ3 levels more than RANKL treatment alone. In conclusion, our data suggest that gingipains augmented the differentiation and function of mature osteoclasts induced by RANKL through the increase in integrin αvβ3.
作为牙龈卟啉单胞菌的关键毒力因子，牙龈蛋白酶在牙周破坏中起着重要作用。本研究旨在探讨牙龈蛋白酶对破骨细胞形成的影响。我们在研究中使用 RAW264.7 细胞作为破骨细胞前体。在实验组中，用牙龈蛋白酶和/或核因子-κB 受体激活剂配体（RANKL）处理细胞。抗酒石酸酸性磷酸酶（TRAP）活性染色试验表明，牙龈蛋白酶呈剂量依赖性增加破骨细胞前体和 RANKL 诱导的成熟破骨细胞。实时逆转录聚合酶链反应分析表明，牙龈蛋白酶上调了破骨细胞基因，包括蛋白酶组织蛋白酶 K（Ctsk）、基质金属蛋白酶 9（Mmp9）、激活 T 细胞核因子 1（Nfatc1）和酸性磷酸酶 5，抗酒石酸（Acp5）呈时间依赖性。Western blot 分析显示，与 RANKL 单独处理相比，牙龈蛋白酶和 RANKL 诱导的 TNF 受体激活因子 6（TRAF6）和整合素β3 表达上调。与对照组相比，FAK 和桩蛋白的磷酸化水平升高，表明整合素相关信号增强。此外，pit 吸收试验表明，牙龈蛋白酶增强了 RANKL 诱导的破骨细胞的骨吸收功能。当我们用西仑吉肽阻断整合素αvβ3 时，与 RANKL 单独处理相比，牙龈蛋白酶逆转了 RANKL 诱导的破骨细胞形成和吸收功能的减少，因为它们增加了整合素αvβ3 的水平超过了 RANKL 处理。总之，我们的数据表明，牙龈蛋白酶通过增加整合素αvβ3 增强了 RANKL 诱导的成熟破骨细胞的分化和功能。
