Department of Pharmacology, Faculty of Pharmacy, Yasuda Women's University, Hiroshima, 731-0153, Japan.
Neurochem Int. 2020 Nov;140:104840. doi: 10.1016/j.neuint.2020.104840. Epub 2020 Aug 25.
Much attention has been paid to the connection between periodontitis and Alzheimer's disease (AD). We previously showed that infection of P. gingivalis, one of major periodontal pathogen causing periodontitis, induced migration and inflammatory responses in murine microglia through gingipain-induced activation of protease-activated receptor 2 (PAR2). In this study, we have attempted to clarify effects of secreted gingipains on cell migration of human microglial cell line, cleavage sites of PAR2 by gingipains and subsequent signaling pathways. P. gingivalis culture supernatant induced migration and membrane ruffling, which is necessary for microglia migration, in human microglial cell line HMC3 cells through PAR2. These effects were mainly mediated by gingipains, because cell migration and membrane ruffling were dramatically inhibited by treatment with gingipain inhibitors. Furthermore, pharmacological and genetic inhibition of Src kinase and β-arrestin, which are important for the internalization of G protein-coupled receptors, significantly inhibited P. gingivavlis culture supernatant-induced membrane ruffling in HMC3 cells. After treatment with P. gingivalis culture supernatant in Flag-PAR2-HA transfected HEK293T cells, Flag was removed from the cell surface, and HA was detected in the cytosol, indicating the internalization of PAR2. Furthermore, the phosphorylation level of ERK1/2 increased in PAR2-transfected HEK293T cells after treatment with P. gingivalis culture supernatant. The gingipain inhibitors, Src kinase inhibitor and β-arrestin knockdown suppressed PAR2 internalization and ERK1/2 phosphorylation. These observations suggest that secreted gingipains from P. gingivalis induce Src- and β-arrestin-dependent internalization of PAR2 and further activate the ERK1/2 pathway to promote migration of microglia. PAR2 are activated by the tethered ligands exposed by cleavage of extracellular N-terminal of PAR2. We also estimated potential gingipain cleavage sites in PAR2 and exposed tethered ligands, which are required for PAR2 internalization and membrane ruffling. The identified mechanism in this study might contribute to the retrogression of sporadic AD in patients after infection with P. gingivalis.
人们高度关注牙周炎和阿尔茨海默病（AD）之间的关联。我们之前曾表明，一种主要的牙周病原体——牙龈卟啉单胞菌（P. gingivalis）的感染会通过牙龈蛋白酶诱导的蛋白酶激活受体 2（PAR2）激活，诱导鼠小胶质细胞的迁移和炎症反应。在这项研究中，我们试图阐明牙龈蛋白酶对人小胶质细胞系细胞迁移的影响、牙龈蛋白酶对 PAR2 的裂解位点以及随后的信号通路。牙龈卟啉单胞菌培养上清液通过 PAR2 诱导人小胶质细胞系 HMC3 细胞的迁移和膜皱襞形成，这是小胶质细胞迁移所必需的。这些效应主要是由牙龈蛋白酶介导的，因为用牙龈蛋白酶抑制剂处理可显著抑制细胞迁移和膜皱襞形成。此外，Src 激酶和β-arrestin 的药理学和遗传学抑制，对于 G 蛋白偶联受体的内化是重要的，可显著抑制 P. gingivavlis 培养上清液诱导的 HMC3 细胞的膜皱襞形成。在用 Flag-PAR2-HA 转染的 HEK293T 细胞中用牙龈卟啉单胞菌培养上清液处理后，Flag 从细胞表面去除，HA 被检测到细胞质中，表明 PAR2 的内化。此外，在牙龈卟啉单胞菌培养上清液处理后，PAR2 转染的 HEK293T 细胞中 ERK1/2 的磷酸化水平增加。牙龈蛋白酶抑制剂、Src 激酶抑制剂和β-arrestin 敲低抑制了 PAR2 的内化和 ERK1/2 的磷酸化。这些观察结果表明，牙龈卟啉单胞菌分泌的牙龈蛋白酶诱导 PAR2 的Src 和β-arrestin 依赖性内化，并进一步激活 ERK1/2 途径促进小胶质细胞的迁移。PAR2 通过 PAR2 细胞外 N 端裂解暴露的连接配体激活。我们还估计了 PAR2 内化和膜皱襞形成所需的 PAR2 中的潜在牙龈蛋白酶裂解位点和暴露的连接配体。本研究中确定的机制可能有助于解释牙龈卟啉单胞菌感染后散发性 AD 患者的退行性变。
