S100A9 Increases IL‐6 and RANKL Expressions through MAPKs and STAT3 Signaling Pathways in Osteocyte‐Like Cells

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.

The presence of neutrophils causes RANKL expression in periodontal tissue, giving rise to osteoclast formation

“…Neutrophils can produce proteins, including S100A9, IL‐1, IL‐6, and TNF, that stimulate RANKL expression 37‐39 . These proteins secreted from neutrophils may increase RANKL expression in osteoblasts, PDL cells, osteocytes, and lymphocytes, resulting in subsequent increased RANKL expression in periodontal tissue.…”

“…Neutrophils can produce proteins, including S100A9, IL-1, IL-6, and TNF, that stimulate RANKL expression. [37][38][39] These proteins secreted from neutrophils may increase RANKL expression in osteoblasts, PDL cells, osteocytes, and lymphocytes, resulting in subsequent increased RANKL expression in periodontal tissue. Air pouch exudates showed increased numbers of CD11b + Ly6G + RANKL + neutrophils following LPS injection and reduced CD11b + Ly6G + RANKL + neutrophil numbers following anti-Ly6G Ab administration in LPSinjected air pouches.…”

PAMPs and DAMPs as the Bridge Between Periodontitis and Atherosclerosis: The Potential Therapeutic Targets

“…Immunohistochemistry and RNA sequencing showed that S100A8 and S100A9 were highly expressed in the ligatureinduced PD (Maekawa et al, 2019). After mouse osteocyte-like cells (MLO-Y4-A2) were treated with S100A9, the expression of IL-6 and RANKL increased, and the p38/ERK/STAT3 signaling pathway was activated, indicating that alarmins were involved in periodontal bone destruction (Takagi et al, 2020). It is worth noting that the levels of S100A12 and C-reactive protein in the gingival crevicular fluid and serum of patients with PD are elevated and positively correlated with periodontal parameters (Pradeep et al, 2014), suggesting that alarmins may be the link between PD and AS.…”

Biosensor and Lab-on-a-chip Biomarker-identifying Technologies for Oral and Periodontal Diseases

“…S100A8 and S100A9, two proinflammatory calcium-binding S100 proteins acting as damageassociated molecular patterns, were implicated in pathogenesis of rheumatic diseases having strong associations with the disease activity and potential as therapeutic targets (Austermann et al, 2018). S100A9 was also reported to increase IL-6 production and RANKL expression in osteocyte-like cells suggesting potential as osteoimmune biomarker for inflammatory osteolysis (Takagi et al, 2020).…”