Regulation of Inflammatory Markers via PARs in Gingival Epithelial Cells

Protease Activated Receptors (PARs) are a family of G-protein coupled receptors present in gingival epithelial cells (GEC). These receptors can be activated by proteases secreted from major periodontal pathogens. However, little is known about how signaling through these receptors affect epithelial innate immune responses.

Objectives: We investigated how various inflammatory cytokines and receptors are regulated by PAR1 and PAR2 activation. Furthermore, downstream signaling pathways involved in induction of selected markers in innate immunity, CCL20/MIP-3a, CXCL5/ENA-78 and CXCL3/MIP-2b have been studied.

Methods: GECs were stimulated with thrombin and trypsin to activate PAR1 and PAR2, respectively. For gene silencing, we used siRNA to target the human PAR1 or PAR2 gene. Scrambled non-silencing RNA served as a negative control. We used PCR array to study the role of thrombin and trypsin and more specifically PAR1 and PAR2 in induction of inflammatory cytokines and receptors and validated our data by real-time PCR. Downstream signaling was investigated with NFkB inhibitor, JSH-23, phospholipase C inhibitor, U73122 and its control substance, U73343.

Results: Among 84 genes tested, only CXCL3 induction by thrombin was via PAR1 while CXCL3, CXCL5 and CCL20 induction by trypsin was via PAR2. NFkB inhibitor completely blocked CXCL3 induction by thrombin and CXCL3, CXCL5 and CCL20 induction by tryspin. The PLC inhibitor, compared to its control substance, resulted in partially decreased CXCL3 and CCL20 induction by PAR1 and PAR2, respectively, but no effect on CXCL3 and CXCL5 induction by PAR2.

Conclusion: Our data suggest that PAR2 has a more dramatic effect than PAR1 on GEC expression of inflammatory markers and that NFkB is involved in downstream signaling of PARs in gingival epithelial responses. Our data pave the way for better understanding of the role of PAR signaling on innate immunity.

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