Department of Periodontology and Endodontology, Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima 770-8504, Japan.
Department of Oral Microbiology, Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima 770-8504, Japan.
Biomed Res Int. 2020 Feb 19;2020:7149408. doi: 10.1155/2020/7149408. eCollection 2020.
Calprotectin is a heterocomplex of S100A8 and S100A9 and is mainly secreted from neutrophils, monocytes, and chondrocytes in inflammatory condition. Calprotectin binds to RAGE and TLR4 and induces the expression of proinflammatory chemokines and cytokines in various cells. Periodontitis is a chronic inflammatory disease that leads to gingival inflammation and alveolar bone resorption. Calprotectin levels in gingival crevicular fluid of periodontitis patients are higher than healthy patients. In the present study, the effects of S100A8 and S100A9 on the expressions of proinflammatory cytokines and bone metabolism-related factors in mouse osteocyte-like cells (MLO-Y4-A2) were investigated.
MLO-Y4-A2 cells were treated with S100A8 and S100A9, and the expressions of RAGE, TLR4, RANKL, and several inflammatory cytokines were analyzed by PCR and Western blotting or ELISA methods. To investigate the intracellular signaling pathways, phosphorylation of MAPK and STAT3 was determined by Western blotting, and chemical specific inhibitors and siRNAs were used.
Expressions of IL-6 and RANKL were increased by treatment with S100A9 but not S100A8. However, both S100A8 and S100A9 did not change expression of IL-1, IL-8, and TNF-. Although RAGE and TLR4 expressions were not upregulated by S100A9 treatment, transfection of siRNA for RAGE and TLR4 significantly decreased IL-6 and RANKL expressions. In addition, S100A9 activated p38, ERK, and STAT3 signaling pathways, and inhibitors for these factors significantly decreased S100A9-induced IL-6 and RANKL expressions.
These results indicated that S100A9 induces IL-6 and RANKL production via engagement with RAGE and TLR4 signalings in osteocytes and suggested that S100A9 may play important roles in the periodontal alveolar bone destruction.
钙卫蛋白是 S100A8 和 S100A9 的异源复合物，主要在炎症条件下从中性粒细胞、单核细胞和软骨细胞中分泌。钙卫蛋白与 RAGE 和 TLR4 结合，并在各种细胞中诱导促炎趋化因子和细胞因子的表达。牙周炎是一种导致牙龈炎症和牙槽骨吸收的慢性炎症性疾病。牙周炎患者龈沟液中的钙卫蛋白水平高于健康患者。在本研究中，研究了 S100A8 和 S100A9 对小鼠成骨细胞样细胞（MLO-Y4-A2）中促炎细胞因子和骨代谢相关因子表达的影响。
用 S100A8 和 S100A9 处理 MLO-Y4-A2 细胞，通过 PCR 和 Western blot 或 ELISA 方法分析 RAGE、TLR4、RANKL 和几种炎症细胞因子的表达。为了研究细胞内信号通路，通过 Western blot 测定 MAPK 和 STAT3 的磷酸化，并用化学特异性抑制剂和 siRNA 进行研究。
S100A9 处理可增加 IL-6 和 RANKL 的表达，但 S100A8 则不能。然而，S100A8 和 S100A9 均未改变 IL-1、IL-8 和 TNF-α的表达。尽管 S100A9 处理未上调 RAGE 和 TLR4 的表达，但 RAGE 和 TLR4 的 siRNA 转染可显著降低 IL-6 和 RANKL 的表达。此外，S100A9 激活了 p38、ERK 和 STAT3 信号通路，这些因子的抑制剂可显著降低 S100A9 诱导的 IL-6 和 RANKL 的表达。
这些结果表明，S100A9 通过与成骨细胞中的 RAGE 和 TLR4 信号转导结合，诱导 IL-6 和 RANKL 的产生，并提示 S100A9 可能在牙周牙槽骨破坏中起重要作用。
