Protease-Activated Receptor 2 Mediates Human Beta-Defensin 2 and CC Chemokine Ligand 20 mRNA Expression in Response to Proteases Secreted byPorphyromonas gingivalis

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Abstract
The oral pathogenPorphyromonas gingivalissecretes proteases such as Arg-gingipain B (RgpB) that activate protease-activated receptors (PARs). Human beta-defensins (hBDs) and the macrophage inflammatory protein 3α/CC chemokine ligand 20 (CCL20) produced by epithelial cells are antimicrobial peptides that provide cytokine function and play an important role in innate immunity. The aim of the present study was to determine whether specific members of the PAR family mediate the expression of these innate immunity markers in gingival epithelial cells (GECs) when exposed toP. gingivaliscell-free culture supernatant or purified RgpB. hBD-2 mRNA in GECs was induced in response to supernatant and purified RgpB fromP. gingivalis(P= 0.02 andP= 0.016, respectively). This effect was abrogated by the protease inhibitor tosyl-l-lysine chloromethyl ketone (TLCK) (P< 0.05). In response toP. gingivalissupernatant and to purified RgpB, the hBD-2 mRNA expression was significantly decreased in PAR-2 gene knockdown cells, whereas no change was detected in PAR-1 gene knockdown cells. CCL20 mRNA expression also increased in response to the supernatant ofP. gingivalis, and this effect was blocked by the protease inhibitor, TLCK (P= 0.05 andP= 0.024, respectively), and was blocked in PAR-2 gene knockdown cells. Our data indicate that hBD-2 and CCL20 mRNA up-regulation byP. gingivalissupernatant and purified RgpB was mediated via PAR-2, but not via PAR-1, and that proteases play a role in the regulation of innate immune responses in GECs. GECs use PARs to recognizeP. gingivalisand mediate cell responses involved in innate immunity.