Department of Periodontology, Peking University School and Hospital of Stomatology, Beijing, China.
Department of Periodontology, School and Hospital of Stomatology, Tianjin Medical University, Tianjin, China.
J Periodontal Res. 2017 Dec;52(6):975-983. doi: 10.1111/jre.12465. Epub 2017 Jun 23.
Calprotectin (S100A8/A9) is a heterodimer of S100A8 and S100A9 and is associated with multiple inflammatory diseases, including Crohn's disease, rheumatoid arthritis and periodontitis. Levels of calprotectin are elevated in the gingival crevicular fluid of patients with periodontitis; however, the effects of calprotectin on human gingival fibroblasts (HGFs) remain unknown. This study investigated the proinflammatory activity of calprotectin on HGFs and the functional receptors and signaling pathways engaged by calprotectin.
HGFs were stimulated by equimolar concentrations of S100A8 and/or S100A9, and the expression levels of interleukin (IL)-6 and IL-8 were detected using real-time quantitative polymerase chain reaction and enzyme-linked immunosorbent assays. The calprotectin receptors were identified by pre-incubating HGFs with the toll-like receptor (TLR) 4 inhibitor or the antibody targeting the advanced glycation end product receptor (RAGE). The involvement of reactive oxygen species (ROS) and signaling pathways were also investigated by treating HGFs with ROS inhibitor or specific pathway inhibitors, respectively.
S100A9 and S100A8/A9 significantly upregulated IL-6 and IL-8 expression, which was inhibited upon treatment with the TLR4 inhibitor TAK242. Pretreatment with RAGE-blocking antibodies did not affect cytokine expression. Additionally, S100A9 promoted the production of IL-6 and IL-8 from HGFs via different signaling pathways. IL-6 expression was upregulated via the NF-κB, c-Jun amino-terminal kinase (JNK) 1/2 and p38 mitogen-activated protein kinase (MAPK) pathways, and IL-8 expression was upregulated via NF-κB, p38, JNK1/2 and extracellular-regulated kinase 1/2 MAPK pathways. The release of both cytokines was dependent upon the production of ROS.
Our findings suggest that calprotectin exerts proinflammatory effects on HGFs via the S100A9 subunit and TLR4-mediated NF-κB and MAPK signaling pathways.
钙卫蛋白（S100A8/A9）是 S100A8 和 S100A9 的异二聚体，与多种炎症性疾病相关，包括克罗恩病、类风湿关节炎和牙周炎。牙周炎患者的龈沟液中钙卫蛋白水平升高；然而，钙卫蛋白对人牙龈成纤维细胞（HGFs）的影响尚不清楚。本研究探讨了钙卫蛋白对 HGFs 的促炎活性以及钙卫蛋白结合的功能受体和信号通路。
用等摩尔浓度的 S100A8 和/或 S100A9 刺激 HGFs，并用实时定量聚合酶链反应和酶联免疫吸附试验检测白细胞介素（IL）-6 和 IL-8 的表达水平。通过用 toll 样受体（TLR）4 抑制剂或针对晚期糖基化终产物受体（RAGE）的抗体预先孵育 HGFs，鉴定钙卫蛋白受体。还分别用活性氧（ROS）抑制剂和特定途径抑制剂处理 HGFs，研究 ROS 和信号通路的参与情况。
S100A9 和 S100A8/A9 显著上调了 IL-6 和 IL-8 的表达，TLR4 抑制剂 TAK242 处理后抑制了这一表达。用 RAGE 阻断抗体预处理并不影响细胞因子的表达。此外，S100A9 通过不同的信号通路促进 HGFs 产生 IL-6 和 IL-8。IL-6 的表达通过 NF-κB、c-Jun 氨基末端激酶（JNK）1/2 和 p38 丝裂原激活蛋白激酶（MAPK）途径上调，IL-8 的表达通过 NF-κB、p38、JNK1/2 和细胞外调节激酶 1/2 MAPK 途径上调。两种细胞因子的释放都依赖于 ROS 的产生。
我们的研究结果表明，钙卫蛋白通过 S100A9 亚基和 TLR4 介导的 NF-κB 和 MAPK 信号通路对 HGFs 发挥促炎作用。
