The role of PAR1 and PAR2 in gingival inflammatory responses

Protease activated receptors (PARs) are a family of four G-protein-coupled receptors and have various functions on different cell types. Although PAR1 and PAR2 are highly expressed on gingival epithelial cells (GECs), there is little information about their role in GECs. Understanding these receptors is important as many periodontal pathogens have proteases as part of their virulence factors.

Objectives: Following up on our previous microarray data, we investigated the role of PAR1 vs. PAR2 in the induction of two inflammatory markers: CCL20/MIP3a, with antimicrobial properties and dendritic cell attraction, and CXCL5/ENA-78, an epithelial neutrophil attractant.

Methods: GECs and hTERT cells, an immortalized oral epithelial cell line, were stimulated with thrombin (0.003-30 U/ml) and trypsin (1-20 nM) to activate PAR1 and PAR2, respectively, for 6 hours. PPACK and TLCK were used as inhibitors of thrombin and trypsin, respectively. Gene expression levels were analyzed with real-time PCR. For further analysis, siRNAs specific for PAR1 and PAR2 were utilized to block expression of each receptor, and a mixture of ATAP2/WEDE15 antibodies was used to inhibit PAR1 cleavage. All data are from three independent experiments with duplicate samples.

Results: Dose-dependent responses for CCL20 and CXCL5 mRNA expression were observed upon activation of PAR1 and PAR2 in both primary and immortalized cells. In all experiments the effects of thrombin and trypsin were abrogated by the protease inhibitors. CXCL5 induction by trypsin was completely inhibited in cells transfected with PAR2 siRNA, while CCL20 induction by trypsin was partially blocked by PAR2 siRNA. However, antibody blocking showed a role for PAR1 in CXCL5 but not in CCL20 up-regulation by thrombin.

Conclusion: Our data suggest PAR2 mediates CCL20 and CXCL5 up-regulation; while PAR1 may mediate CXCL5 up-regulation, but not CCL20. How PARs mediate inflammatory responses in GECs will lead to better understanding of epithelial innate immunity.

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