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Gingipains promote RANKL-induced osteoclastogenesis through the enhancement of integrin β3 in RAW264.7 cells.
Journal of Molecular Histology ( IF 3.2 ) Pub Date : 2020-03-19 , DOI: 10.1007/s10735-020-09865-w
Weiyan Mo 1, 2 , Haoyuan Luo 1, 2 , Juan Wu 3 , Na Xu 4 , Fuping Zhang 1, 2 , Qihong Qiu 1, 2 , Wenjun Zhu 1, 2 , Min Liang 1, 2
Affiliation  

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.

中文翻译:

姜黄素通过增强RAW264.7细胞中的整合素β3来促进RANKL诱导的破骨细胞生成。

作为牙龈卟啉单胞菌的重要毒力因子,牙龈蛋白酶在牙周破坏中起重要作用。这项研究旨在调查姜黄素对破骨细胞形成的影响。在我们的研究中,我们将RAW264.7细胞用作破骨细胞前体。在实验组中,用牙龈蛋白酶和/或核因子-κB配体的受体激活剂(RANKL)处理细胞。抗酒石酸酸性磷酸酶(TRAP)活性染色法显示破骨细胞前体和RANKL诱导的成熟破骨细胞以姜黄素剂量依赖性方式增加。实时逆转录聚合酶链反应分析表明,姜黄素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9(Mmp9),活化T细胞1(Nfatc1)和酸性磷酸酶5的酒石酸抗性(Acp5)以时间相关的方式进行。Western blotting检测显示与单独的RANKL相比,由姜黄素和RANKL诱导的TNF受体激活因子6(TRAF6)和整联蛋白β3的表达上调。与对照相比,FAK和paxillin的磷酸化升高也证明了整合素相关信号的增强。此外,凹坑吸收试验表明,姜黄素增强了RANKL诱导的破骨细胞的骨吸收功能。当我们使用Cilengitide阻断整联蛋白αvβ3时,姜黄素逆转了RANKL诱导的破骨细胞的形成和吸收功能的降低,因为它们比单独RANKL治疗更能增强整联蛋白αvβ3的水平。总之,我们的数据表明,姜黄素通过整合素αvβ3的增加增强了RANKL诱导的成熟破骨细胞的分化和功能。
更新日期:2020-03-19
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