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.

牙周炎与阿尔茨海默病 (AD) 之间的联系备受关注。我们之前表明，牙龈卟啉单胞菌是引起牙周炎的主要牙周病原体之一，通过牙龈蛋白酶诱导的蛋白酶激活受体 2 (PAR2) 激活，在小鼠小胶质细胞中诱导迁移和炎症反应。在这项研究中，我们试图阐明分泌型牙龈蛋白酶对人小胶质细胞系细胞迁移、牙龈蛋白酶对 PAR2 的裂解位点和随后的信号通路的影响。牙龈卟啉单胞菌培养上清液通过 PAR2 在人小胶质细胞系 HMC3 细胞中诱导小胶质细胞迁移所必需的迁移和膜皱褶。这些作用主要由 gingipains 介导，因为用 gingipain 抑制剂处理显着抑制了细胞迁移和膜皱褶。此外，对 G 蛋白偶联受体的内化很重要的 Src 激酶和 β-抑制蛋白的药理学和遗传抑制显着抑制了牙龈卟啉单胞菌培养物上清液诱导的 HMC3 细胞膜褶皱。用牙龈卟啉单胞菌治疗后Flag-PAR2-HA转染的HEK293T细胞培养上清液，从细胞表面去除Flag，胞质溶胶中检测到HA，表明PAR2内化。此外，在用牙龈卟啉单胞菌培养上清液处理后，在 PAR2 转染的 HEK293T 细胞中 ERK1/2 的磷酸化水平增加。gingipain 抑制剂、Src 激酶抑制剂和 β-arrestin 敲低抑制了 PAR2 内化和 ERK1/2 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-抑制蛋白依赖性内化，并进一步激活 ERK1/2 通路以促进小胶质细胞的迁移。PAR2 由通过裂解 PAR2 的细胞外 N 端而暴露的系留配体激活。我们还估计了 PAR2 中潜在的牙龈蛋白酶裂解位点和暴露的系留配体，这是 PAR2 内化和膜褶皱所必需的。本研究中确定的机制可能有助于患者感染牙龈卟啉单胞菌后散发性 AD 的倒退。