Arginine-specific protease from Porphyromonas gingivalis activates protease-activated receptors on human oral epithelial cells and induces interleukin-6 secretion

Periodontitis is a chronic inflammatory disease affecting oral tissues. Ora l epithelial cells represent the primary barrier against bacteria causing t he disease. We examined the responses of such cells to an arginine-specific cysteine proteinase (RgpB) produced by a causative agent of periodontal di sease, Porphyromonas gingivalis. This protease caused an intracellular calc ium transient in an oral epithelial cell line (KB), which was dependent on its enzymatic activity. Since protease-activated receptors (PARs) might med iate such signaling, reverse transcription-PCR was used to characterize the range of these receptors expressed in the KB cells. The cells were found t o express PAR-1, PAR-2, and PAR-3, but not PAR-4. In immunohistochemical st udies, human gingival epithelial cells were found to express PAR-1, PAR-2, and PAR-3 on their surface, but not PAR-4, indicating that the cell line wa s an effective model for the in vivo situation. PAR-1 and PAR-2 expression was confirmed in intracellular calcium mobilization assays by treatment of the cells with the relevant receptor agonist peptides. Desensitization expe riments strongly indicated that signaling of the effects of RgpB was occurr ing through PAR-1 and PAR-2. Studies with cells individually transfected wi th each of these two receptors confirmed that they were both activated by R gpB. Finally, it was shown that, in the oral epithelial cell line, PAR acti vation by the bacterial protease-stimulated secretion of interleukin-6. Thi s induction of a powerful proinflammatory cytokine suggests a mechanism whe reby cysteine proteases from P. gingivalis might mediate inflammatory event s associated with periodontal disease on first contact with a primary barri er of cells.