PROTEASE-ACTIVATED RECEPTORS AND THEIR ROLE IN IL-6 AND NF-IL-6 EXPRESSION IN HUMAN GINGIVAL FIBROBLASTS

The serine protease thrombin is formed at sites of coagulation and inf lammation and has been shown to have important proinflammatory cellula r effects relevant to the pathogenesis of periodontal disease. Thrombi n acts via specific cell surface receptors termed protease-activated r eceptor-1 (PAR-1) and PAR-3, which have a distinctive method of activa tion. Proteolytic cleavage of the extracellular domain by thrombin rev eals a hidden amino terminus which then acts as a ''tethered ligand''. A short synthetic peptide (SFLLRN) can also mimic the tethered ligand and activate PAR-1 but not PAR-3. Also, a trypsin-sensitive receptor termed PAR-2 has been described which is activated by the PAR-1 activa ting peptide SFLLRN. Here we show conclusively by flow cytometric and Northern blot analysis that human gingival fibroblasts (HGF) express P AR-1 bur not PAR-2. In functional studies we also show that thrombin a nd SFLLRN stimulated increased expression of mRNA encoding nuclear tra nscription factor NF-IL-6 and IL-6 in vitro. At optimal concentrations , thrombin (10(-7) M) induced 7.6 +/- 0.01 ng/ml immunoactive IL-6 and PAR-1 activating peptide (5 x 10(-5) M) induced 2.2+/-0.2 ng/ml (mean +/- standard error of mean). A proteolytically inactive recombinant t hrombin (serine 195 to alanine) was without activity. These data show that HGF express PAR-1 and suggest that PAR-1 activation stimulates in creased NF-IL-6 and IL-6 gene expression and IL-6 secretion by HGF in vitro. Whether HGF express PAR-3 is unknown, but the fact that SFLLRN was not a complete replacement for thrombin raises the possibility tha t HGF may express additional thrombin receptors. These findings add we ight to the importance of the cytokine-like role played by thrombin an d raise the possibility that protease-activated receptors may play a r ole in the pathogenesis of inflammatory periodontal disease.