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

Abstract

Proteinase 3 (PR3), a 29-kDa serine proteinase secreted from activated neutrophils, also exists in a membrane-bound form, and is suggested to actively contribute to inflammatory processes. The present study focused on the mechanism by which PR3 activates human oral epithelial cells. PR3 activated the epithelial cells in culture to produce IL-8 and monocyte chemoattractant protein-1 and to express ICAM-1 in a dose- and time-dependent manner. Incubation of the epithelial cells for 24 h with PR3 resulted in a significant increase in the adhesion to neutrophils, which was reduced to baseline levels in the presence of anti-ICAM-1 mAb. Activation of the epithelial cells by PR3 was inhibited by serine proteinase inhibitors and serum. The epithelial cells strongly express protease-activated receptor (PAR)-1 and PAR-2 mRNA and weakly express PAR-3 mRNA. The expression of PAR-2 on the cell surface was promoted by PR3, and inhibited by cytochalasin B, but not by cycloheximide. PR3 cleaved the peptide corresponding to the N terminus of PAR-2 with exposure of its tethered ligand. Treatment with trypsin, an agonist for PAR-2, and a synthetic PAR-2 agonist peptide induced intracellular Ca2+ mobilization, and rendered cells refractory to subsequent stimulation with PR3 and vice versa. The production of cytokine induced by PR3 and the PAR-2 agonist peptide was completely abolished by a phospholipase C inhibitor. These findings suggest that neutrophil PR3 activates oral epithelial cells through G protein-coupled PAR-2 and actively participates in the process of inflammation such as periodontitis.

N-Linked Glycosylation Regulates Human Proteinase-activated Receptor-1 Cell Surface Expression and Disarming via Neutrophil Proteinases and Thermolysin

“…One previous study on human oral epithelial cells (33) also pointed out that proteinase 3 can rapidly cleave PAR 2 between Arg and Ser and relatively inefficiently cleave between Lys and Val. Furthermore, proteinase 3 cleaved the peptide corresponding to the N terminus of PAR 2 at Arg 36 -Ser 37 , indicating that the site of the PAR 2 is structurally accessible by proteinase 3 (33). Interestingly, one previous study on synthetic peptides (13) 49 .…”

Emerging roles of serine proteinases in tissue turnover in arthritis

“…Thus, increased expression of serine proteinases capable of activating PARs (Figure 1) has the potential to exacerbate inflammation. For example, PAR-2 can be activated by trypsin, mast cell tryptase (163), tissue factor-factor VIIa and factor Xa (15), some kallikreins (164), PR3 (165), and matriptase (166).…”

Physiology and Pathophysiology of Proteinase-Activated Receptors (PARs): PARs in the Respiratory System: Cellular Signaling and Physiological/Pathological Roles

“…Trypsin, tryptase, and coagulation factors VIIa and Xa are considered possible endogenous activators for PAR-2 in the airway (6), although additional novel candidates for PAR-2 agonists including trypsin IV (48), neutrophil proteinase 3 (49), and membrane-type serine protease-1 (50) have been reported. Most interestingly, house dust mite allergens, such as Der p1, Der p3, and Der p9, are capable of activating PAR-2 and stimulating release of pro-inflammatory cytokines including GM-CSF, eotaxin, IL-6, and IL-8 in human lung epithelial cells, although Der p1 might inactivate PAR-1 (51,52).…”