Advanced glycation end-products increase lipocalin 2 expression in human oral epithelial cells.

Background And Objectives: Diabetes mellitus (DM), a risk factor of periodontal diseases, exacerbates the pathological condition of periodontitis. A major factor for DM complications is advanced glycation end-products (AGEs) that accumulate in periodontal tissues and cause inflammatory events. Lipocalin 2 (LCN2) is an antimicrobial peptide and inflammation-related factor, and LCN2 levels increase in DM. In this study, the effects of AGEs and lipopolysaccharide of Porphyromonas gingivalis (P g-LPS) on LCN2 expression in human oral epithelial cells (TR146 cells) and the role of secreted LCN2 in periodontitis with DM were investigated.

Material And Methods: TR146 cells were cultured with AGEs (AGE2) and control BSA and cell viability was estimated, or with P g-LPS. Conditioned medium and cell lysates were prepared from cultures of epithelial cells and used for Western blotting and ELISA to analyze LCN2, RAGE, IL-6, MAPK, and NF-κB. RNA was isolated from AGE-treated TR146 cells and differentiated HL-60 (D-HL-60) cells and used for quantitative real-time PCR to examine the expression of LCN2 and interleukin-6 (IL-6) mRNAs. RAGE- and LCN2-siRNAs (siRAGE, siLCN2) were transfected into epithelial cells, and AGE-induced LCN2 expression was investigated. D-HL-60 cells were co-cultured with TR146 cells that were transfected with siLCN2 and treated with AGEs, and IL-6 mRNA expression in D-HL-60 cells and cell migration was investigated.

Results: AGEs increased the expression levels of LCN2 and IL-6 in oral epithelial cells. siRAGE and a neutralizing antibody for RAGE inhibited AGE-induced LCN2 expression. AGEs stimulated the phosphorylation of ERK, p38, and NF-κB in epithelial cells, and their inhibitors suppressed AGE-induced LCN2 expression. In contrast, P g-LPS did not show a significant increase in LCN2 level in TR146 cells that expressed Toll-like receptor 2. In co-culture experiments, AGE-induced LCN2 inhibited IL-6 mRNA expression in D-HL-60 cells, and LCN2 knockdown in epithelial cells suppressed HL-60 cell migration.

Conclusion: These results suggested that AGEs increase LCN2 expression via RAGE, MAPK, and NF-κB signaling pathways in oral epithelial cells, and secreted LCN2 may influence the pathological condition of periodontitis with DM.