Abstract
Objective 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. Design 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. Results 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-1β, IL-8, and TNF-α. 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. Conclusions 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.
Highlights
S100A8 and S100A9 proteins are subunits of the calciumbinding protein and are abundant in myeloid cells such as neutrophils, monocytes, and chondrocytes [1]. e heterocomplex of S100A8 and S100A9 has been known as calprotectin, which is detected in various tissues
Calprotectin acts in a cytokine-like manner by binding to cell surface receptors: Toll-like receptor 4 (TLR4) and receptor of advanced glycation end-products (RAGE) [5], and induces the expression of various proinflammatory chemokines and cytokines, such as C-X-C motif ligand (CXCL)1, interleukin (IL)-6, IL-8, and tumor necrosis factor (TNF)-α [6, 7]. erefore, it has been considered that calprotectin belongs to damage-associated molecular patterns (DAMPs) or alarmin by increasing the expression of inflammatory mediators in several inflammatory diseases
High levels of calprotectin were detected in gingival crevicular fluid (GCF) from patients with periodontitis, and high levels of S100A8 and S100A9 were detected in blood vessels in Porphyromonas gingivalis (Pg)-infected mice [14, 15]. ese reports suggest that calprotectin expression levels in patients with periodontitis were increased by its causal agents, such as Pg LPS, and plays important roles in periodontal inflammatory responses
Summary
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. The effects of S100A8 and S100A9 on the expressions of proinflammatory cytokines and bone metabolismrelated 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. S100A9 activated p38, ERK, and STAT3 signaling pathways, and inhibitors for these factors significantly decreased S100A9-induced IL-6 and RANKL expressions. Ese 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 Conclusions. ese 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
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