S100A8, S100A9, and calprotectin (the S100A8/S100A9 complex) are calcium-binding proteins that promote extracellular pro-inflammatory functions and may play an important role in periodontal disease. Both toll-like receptor 4 (TLR4) and the receptor for advanced glycation end-products (RAGE) are thought to be important receptors for S1 00A8, Si 00A9, and calprotectin, but the specific pathways in periodontal ligament (PDL) cells are not yet clear. Our study was designed to identify the specific receptors for Si 00A9 in human PDL cells. Additionally, we investigated the specific pathways that activate the secretion of pro-inflammatory cytokines interleukins (IL)-6 and IL-8 in PDL cells. The role of nuclear factor (NF)-kappa B, mitogen-activated protein kinase (MAPK) and reactive oxygen species (ROS) in S100A9-induced proinflammatory cytokines were investigated through western blot analysis, dichlorodihydrofluorescein diacetate (H(2)DCFDA) probe and the application of specific pathway inhibitors. Our results suggest that the S100A9-induced release of IL-6 and IL-8 from human PDL cells is dependent on TLR4, but not RAGE. We provide evidence that S100A9 promotes the secretion of IL-6 and IL-8 through different pathways. Specifically, Si100A9 up-regulates the secretion of IL-6 from human PDL cells through NF-kappa B and p38 pathways and up-regulates the release of IL-8 from human PDL cells through the NF-kappa B, extracellular-regulated kinase (ERK) 1/2, c-Jun amino-terminal kinase UNK) 1/2, and p38 signaling pathways. In addition, the release of both cytokines depends on ROS production. The release of both cytokines depends on ROS production. These results suggest that S100A9 promotes pro-inflammatory responses in PDL cells through the TLR4-mediated NF-kappa B and MAPK signaling pathways. (C) 2015 Elsevier Ltd. All rights reserved.