Response of Oral Epithelial Cells to Protease Activated Receptor Stimulation

Protease activated receptors (PARs) are a family of receptors associated with both inflammatory and protective responses of various cell types. PARs may be important in gingival epithelial cells (GECs) for recognition and response to periodontal pathogens which secrete proteases as part of their virulence factors. Objectives: we investigated the response of GECs to stimulation of PAR1 and PAR2 in order to identify their effects on inflammatory responses. Methods: primary human GECs were stimulated with thrombin (200nM) or agonist peptide, SLIGRL (20 µM), to stimulate PAR1 and PAR2, respectively. Microarray analysis was performed on total RNA of triplicate samples stimulated for 6 or 24 hr. Selected data were confirmed by quantitative PCR. We visualized the change in localization of PAR1 and PAR2 upon activation and evaluated NF-kB p65 nuclear translocation in cells in which PAR1 or PAR2 was activated by immunofluorescence (IF). Results: microarray data show that PAR1 and 2 signal many similar gene expression responses including genes associated with biotic stimuli and inflammatory responses. Activation of both receptors results in rapid upregulation of genes that are neutrophil attractants, CXCL1,2,3,5 and IL8, as well as proinflammatory cytokines IL1a/b. PCR results confirmed upregulation of IL-8, CXCL5 and CCL20. IF confirmed translocation of PAR1 and PAR2 from cell membrane into the cytoplasm upon activation; PAR2 localized in endocytic clusters, while PAR1 localized more diffusely within the cytoplasm. Differences in responses were also observed: PAR2 activation showed changes in expression of genes associated with growth and development. Inhibitors of NF-kB function were more upregulated with PAR1 activation. Interestingly, the NF-kB p65/RelA subunit was rapidly translocated to nuclei with PAR2 activation but not PAR1 activation. Conclusion: we suggest that both PAR1 and PAR2 have distinct functions for signaling epithelial cell responses to proteases released by injury, PMN activation, or bacteria associated with periodontal disease. NIDCR#R21DE01597