Arginine-Specific Protease fromPorphyromonas gingivalisActivates Protease-Activated Receptors on Human Oral Epithelial Cells and Induces Interleukin-6 Secretion

Abstract

Periodontitis is a chronic inflammatory disease affecting oral tissues. Oral epithelial cells represent the primary barrier against bacteria causing the disease. We examined the responses of such cells to an argininespecific cysteine proteinase (RgpB) produced by a causative agent of periodontal disease, Porphyromonas gingivalis. This protease caused an intracellular calcium transient in an oral epithelial cell line (KB), which was dependent on its enzymatic activity. Since protease-activated receptors (PARs) might mediate such signaling, reverse transcription-PCR was used to characterize the range of these receptors expressed in the KB cells. The cells were found to express PAR-1, PAR-2, and PAR-3, but not PAR-4. In immunohistochemical studies, 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 was 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 experiments strongly indicated that signaling of the effects of RgpB was occurring through PAR-1 and PAR-2. Studies with cells individually transfected with each of these two receptors confirmed that they were both activated by RgpB. Finally, it was shown that, in the oral epithelial cell line, PAR activation by the bacterial protease-stimulated secretion of interleukin-6. This induction of a powerful proinflammatory cytokine suggests a mechanism whereby cysteine proteases from P. gingivalis might mediate inflammatory events associated with periodontal disease on first contact with a primary barrier of cells.

Activation of Human Oral Epithelial Cells by Neutrophil Proteinase 3 Through Protease-Activated Receptor-2

“…Oral epithelial cells in culture strongly expressed PAR-1 and PAR-2 mRNA, weakly expressed PAR-3 mRNA, and did not express PAR-4 mRNA, as assessed by RT-PCR analysis (Fig. 4A), which was consistent with the findings of immunohistochemistry using sections of human gingival tissues (9). By flow cytometric analyses, weak expression of PAR-2 was detected on the untreated cell surface, but the expression of PAR-1 and -3 on the cell surface was below the detectable limit (Fig.…”

“…Three of them (PAR-1, PAR-3, and PAR-4) are activated mainly by thrombin; the fourth (PAR-2) is activated by other serine proteinases such as trypsin, tryptase, and factor Xa (1)(2)(3). It is reported that more than one family member can be present in the same cell: human platelets express PAR-1 and PAR-4 (4, 5); mouse platelets express PAR-3 and PAR-4 (6); human endothelial cells express PAR-1, PAR-2, and possibly PAR-3 (7,8); and human oral epithelial cells express PAR-1, PAR-2, and PAR-3 (9). PAR-1 is the most widely expressed receptor among PAR family members in humans and mice, and it is reported that PAR-1 on human platelets and endothelial cells is inactivated by neutrophil serine proteinases, human leukocyte elastase (HLE), cathepsin G (Cat G), and proteinase 3 (PR3), by cleavage downstream of the tethered ligand (10).…”

Mold Allergen, Pen c 13, Induces IL-8 Expression in Human Airway Epithelial Cells by Activating Protease-Activated Receptor 1 and 2

“…The effects of these aeroallergen proteases, acting via protease-activated receptors (PARs), 3 have been implicated in the regulation of the release of proinflammatory cytokines, such as eotaxin, GM-CSF, IL-6, and IL-8, by epithelial cells in a nonallergic inflammatory response (1)(2)(3)(4)(5)(6)(7)(8)(9)(10). PARs appear to play a proinflammatory role by activating proinflammatory cytokines (11,12). A variety of proinflammatory cytokines are released from the airway epithelium, all of which have the potential to contribute to respiratory diseases, such as asthma, bronchiectasis, and chronic obstructive pulmonary diseases (13,14).…”

Possible Roles of IL-33 in Periodontal Diseases: Porphyromonas gingivalis Induced IL-33 in Human Gingival Epithelial Cells

“…Rgps are capable of activating PAR-2 expressed on human gingival epithelial cells, which produce proinflammatory cytokines [21]. We examined the possible involvement of PAR-2 in P. gingivalisinduced IL-33 mRNA expression.…”