Porphyromonas gingivalis Secreted Proteases Induce Defensin Gene Expression via PAR-2

Objectives: Human beta-defensins (hBDs) are antimicrobial peptides produced by epithelial cells, and they play an important role in innate immunity. Porphyromonas gingivalis, involved in the development of periodontitis, secretes proteases (gingipains) that are important virulence factors. These gingipains cleave protease-activated receptors (PARs) associated with platelet aggregation and immune responses. The aim of this study was to determine whether gene expression of hBD-2 and other markers of innate immunity is regulated via PAR-1 or PAR-2 when exposed to enzymes secreted by P. gingivalis. Methods: Human gingival epithelial cells (GECs) were transfected with small inhibitory RNA (siRNA) targeting the PAR-1 and PAR-2 genes. Cells were stimulated with cell-free supernatant from P. gingivalis for 16 hours. The gene expression of PAR-1, PAR-2, hBD-2, IL-8, and CCL20 was determined using real-time PCR. PAR-1 and PAR-2 localization in cells was monitored by immunofluorescence. Results: hBD-2 mRNA expression in GECs was induced in response to supernatant from P. gingivalis. The effect of the supernatant was abrogated by the used of the protease inhibitor, TLCK. In response to supernatant from P. gingivalis, the hBD-2 mRNA expression was significantly decreased (p<0.05) in PAR-2 gene knock-down cells, but increased in PAR-1 gene knock-down cells. CCL20 and IL-8 gene expression also increased in response to the supernatant and blocked by TLCK demonstrating the role of proteases, but expression was dependent on both PAR-1 and PAR-2 receptors. Immunofluorescence showed internalization of PAR-2 receptor into the cytoplasm of GECs after 30 min exposure to bacterial supernatant. Conclusion: Proteases synthesized by P. gingivalis play a role in the regulation of the hBD-2, CCL20, and IL-8 gene expression in GECs. hBD-2 mRNA up-regulation was mediated via PAR-2, but not via PAR-1. Gingival epithelial cells use PARs to recognize P. gingivalis and mediate cell responses involved in innate immunity. Supported by NIDCR R01DE16961.