Critical roles of PU.1/cathepsin S activation in regulating inflammatory responses of macrophages in periodontitis.

ABSTRACT

OBJECTIVE: To determine the critical roles of PU.1/cathepsin S activation in regulating inflammatory responses of macrophages during periodontitis. BACKGROUND: Cathepsin S (CatS) is a cysteine protease and exerts important roles in the immune response. Elevated CatS has been found in the gingival tissues of periodontitis patients and is involved in alveolar bone destruction. However, the underlying mechanism of CatS-driven IL-6 production in periodontitis remains unclear. METHODS: Western blot was applied to measure mature cathepsin S(mCatS) and IL-6 expression in gingival tissues from periodontitis patients and RAW264.7 cells exposed to lipopolysaccharide from Porphyromonas gingivalis (P.g. LPS). Immunofluorescence was applied to confirm the localization of PU.1, and CatS in the gingival tissues of periodontitis patients. ELISA was performed to determine IL-6 production by the P.g. LPS-exposed RAW264.7 cells. Knockdown by shRNA was used to determine the effects of PU.1 on p38/ nuclear factor (NF)-κB activation, mCatS expression and IL-6 production in RAW264.7 cells. RESULTS: The expressions mCatS and IL-6 were significantly upregulated in gingival macrophages. In cultured RAW264.7 cells, increased mCatS and IL-6 protein paralleled the activation of p38 and NF-κB after exposure to P.g. LPS. CatS knockdown by shRNA significantly decreased P.g. LPS-induced IL-6 expression and p38/NF-κB activation. PU.1 was significantly increased in P.g. LPS-exposed RAW264.7 cells, and PU.1 knockdown dramatically abolished the P.g. LPS-induced upregulation of mCatS and IL-6 and the activation of p38 and NF-κB. Furthermore, PU.1 and CatS colocalized in macrophages within the gingival tissues of periodontitis patients. CONCLUSION: PU.1-dependent CatS drives IL-6 production in macrophages by activating p38 and NF-κB in periodontitis.