Upregulation of Beta-defensins by Mechanical Stress in Keratinocytes via MAPkinase

Objectives: Periodontal tissues are exposed to mechanical forces during the act of mastication. The effect of mechanical stress on the mesenchymal cells derived from periodontal ligament and bone has been well documented. Little, however, is known about the effect of mechanical stresses on the keratinocytes. Human beta-defensins (hBDs) belong to a group of antimicrobial peptides expressed in keratinocytes, which play an important role in the protective barrier of oral epithelium. The present study investigated the effect of mechanical stress on hBDs expression in keratinocyte. Methods: HaCaT cells, a human keratinocyte cell line, were cultured in 60 mm culture dishes with DMEM and 10% fetal bovine serum. In order to apply mechanical stress (gravity loading), the culture dishes were centrifuged at 170xg for 10, 30, 60, 90min in a CO₂ incubator. Expression of hBD-1, -2 and -3 in HaCaT cells were observed by RT-PCR and quantitative RT-PCR using TaqMan probes (Applied Biosystem, CA). The phosphorylation of MAP kinases was detected by western blotting with p-p38 and p-ERK antibodies (SantaCruz,CA). Several experiments were performed, each in triplicate. The data was analyzed using one-way ANOVA. Differences between experimental groups were considered statistically significant at the p<0.05 levels. Results: Expression level of hBD-1 and -3 was significantly higher in the cells in which mechanical stress was applied for 60min than in the control cells (p<0.01). No significant difference was observed in the expression level of hBD-2 between the mechanical stress and control groups. The mechanical stress applied to the cells induced phospholyration of p38 and ERK MAP kinase within 30min. Conclusion: These results indicate that mechanical stress may induce upregulation of hBD-1 and -3 in keratinocyte, and is possibly involved in MAP kinase pathway.