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 inflammation and has been shown to have important proinflammatory cellular effects relevant to the pathogenesis of periodontal disease. Thrombin acts via specific cell surface receptors termed protease-activated receptor-1 (PAR-1) and PAR-3, which have a distinctive method of activation. Proteolytic cleavage of the extracellular domain by thrombin reveals 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 activating peptide SFLLRN. Here we show conclusively by flow cytometric and Northern blot analysis that human gingival fibroblasts (HGF) express PAR-1 but not PAR-2. In functional studies we also show that thrombin and SFLLRN stimulated increased expression of mRNA encoding nuclear transcription 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 thrombin (serine 195 to alanine) was without activity. These data show that HGF express PAR-1 and suggest that PAR-1 activation stimulates increased 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 that HGF may express additional thrombin receptors. These findings add weight to the importance of the cytokine-like role played by thrombin and raise the possibility that protease-activated receptors may play a role in the pathogenesis of inflammatory periodontal disease.

Hou L ，Ravenall S ，Macey MG ，Harriott P ，Kapas S ，Howells GL ... -  《JOURNAL OF PERIODONTAL RESEARCH》

Neutrophil serine proteinases activate human nonepithelial cells to produce inflammatory cytokines through protease-activated receptor 2.

Protease-activated receptors (PARs) compose a family of G protein-coupled receptors activated by proteolysis with exposure of their tethered ligand. Recently, we reported that a neutrophil-derived serine proteinase, proteinase 3 (PR3), activated human oral epithelial cells through PAR-2. The present study examined whether other neutrophil serine proteinases, human leukocyte elastase (HLE), and cathepsin G (Cat G) activate nonepithelial cells, human gingival fibroblasts (HGF). HLE and Cat G as well as PR3 activated HGF to produce IL-8 and monocyte chemoattractant protein 1. Human oral epithelial cells but not HGF express mRNA and protein of secretory leukocyte protease inhibitor, an inhibitor of HLE and Cat G, and recombinant secretory leukocyte protease inhibitor clearly inhibited the activation of HGF induced by HLE and Cat G but not by PR3. HGF express PAR-1 and PAR-2 mRNA in the cells and the proteins on the cell surface. HLE and Cat G 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 Ca(2+) mobilization and rendered cells refractory to subsequent stimulation with HLE and Cat G. The production of cytokine induced by HLE and Cat G and the PAR-2 agonist peptide was completely abolished by inhibition of phospholipase C. These findings suggest that neutrophil serine proteinases have equal ability to activate human nonepithelial cells through PAR-2 to produce inflammatory cytokines and may control a number of inflammatory processes such as periodontitis.

Uehara A ，Muramoto K ，Takada H ，Sugawara S ... -  《JOURNAL OF IMMUNOLOGY》

Thrombin-induced Ca2+ mobilization in human gingival fibroblasts is mediated by protease-activated receptor-1 (PAR-1).

By reverse transcription (RT)-PCR analyses, human gingival fibroblasts (HGFs) were demonstrated to express mRNAs for protease-activated receptor-1 (PAR-1) and PAR-3, although the expression of PAR-3 was much weaker than that of PAR-1. The mRNAs for PAR-2 and PAR-4 were not found by RT-PCR. Furthermore, PAR activation was studied by monitoring cytosolic Ca(2+) concentration ([Ca(2+)]i) in cultured HGFs loaded with fura-2. At concentrations > 0.1 nM, alpha-thrombin caused a transient increase in [Ca(2+)]i in a concentration-dependent manner, and the maximum response was obtained with 10 nM alpha-thrombin. After the [Ca(2+)]i response, the HGFs were completely desensitized to the second stimulation with alpha-thrombin. The PAR-1 agonist peptide SFLLRN produced approximately the same transient [Ca(2+)]i response as alpha-thrombin. After stimulation with SFLLRN, the HGFs did not respond to alpha-thrombin, indicating that treatment with SFLLRN results in complete desensitization to alpha-thrombin. The PAR-2 and PAR-4 agonist peptides had no effect on [Ca(2+)]i in HGFs. These results suggest that alpha-thrombin-induced Ca(2+) mobilization in HGFs is solely mediated by PAR-1.

Tanaka N ，Morita T ，Nezu A ，Tanimura A ，Mizoguchi I ，Tojyo Y ... -  《LIFE SCIENCES》

Thrombin-stimulated growth, clustering, and collagen lattice contraction of human gingival fibroblasts is associated with its protease activity.

Thrombin is a serine protease produced following gingival tissue injury or inflammation. It regulates the functional behavior of injury-neighboring cells via the activation of specific protease-activated receptors (PAR). Thrombin's role in gingival tissue healing and inflammatory response processes is not yet well understood. We investigated the effects of thrombin on gingival fibroblast (GF) growth [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay], collagen lattice contraction, and associated morphological changes. Thrombin (>1 U/ml), but not thrombin receptor (PAR-1) agonist peptide (SFLLRN, single letter amino acid code, abbreviated as TRAP, 1 to 50 microg/ml), stimulated the growth and clustering of cultured human GF in vitro. Growth-stimulatory effects of thrombin were inhibited by D-Phe-Pro-ArgCH2Cl (PPACK), a serine protease inhibitor. By contrast, trypsin (>10 microg/ml), a PAR-2 activator, suppressed the growth of GF. Thrombin (>0.2 U/ml) and TRAP (10 to 25 microg/ml), but not trypsin, prostaglandin E2 (0.01 to 0.5 microg/ml), or bovine serum albumin (BSA) (1 to 80 microg/ml), induced the GF-populated collagen lattice contraction within 30 to 60 minutes of exposure. The thrombin-induced collagen lattice contraction was inhibited by PPACK (20 microg/ml) and an actin filament polymerization inhibitor, cytochalasin B (1 microg/ml). The collagen lattice contraction induced by TRAP was also inhibited by cytochalasin B, but not by PPACK. Using a reverse-transcriptase polymerase chain reaction (RT-PCR), the expression of PAR-1, and to a lesser extent PAR-3, was observed for human GF, although little PAR-2 and PAR-4 expression was noted. These results indicate that thrombin is important in periodontal wound healing and inflammatory processes by promoting the growth and contraction of GF. The stimulatory effects of thrombin are associated with its protease activation of thrombin receptors.

Chang MC ，Chan CP ，Wu HL ，Chen RS ，Lan WH ，Chen YJ ，Jeng JH ... -  《JOURNAL OF PERIODONTOLOGY》

Signaling mechanisms involved in protease-activated receptor-1-mediated interleukin-6 production by human gingival fibroblasts.

Human gingival fibroblasts (HGFs) express protease-activated receptor-1 (PAR-1) at high levels. In cultured HGFs, we studied the signaling pathway of thrombin-induced interleukin-6 (IL-6) production. The PAR-1 agonist peptide SFLLRN mimicked the thrombin-induced IL-6 production in the presence of amastatin, an aminopeptidase inhibitor. Thrombin or a combination of SFLLRN and amastatin also strikingly induced the expression of IL-6 mRNA. Although continuous exposure of HGFs to thrombin rapidly desensitized Ca(2+) signaling, the cells did not lose their ability to produce IL-6 in response to thrombin. Similarly, although treatment of HGFs with BAPTA-AM [1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester], an intracellular Ca(2+) chelator, markedly attenuated the thrombin-induced increase in intracellular Ca(2+) concentration, the same treatment did not suppress the thrombin-induced IL-6 production. However, thrombin-induced IL-6 production was strongly inhibited by the p38 mitogen-activated protein (MAP) kinase and tyrosine kinase inhibitors, and Western blotting analyses showed that thrombin stimulates p38 MAP kinase phosphorylation. Specific inhibitors that inhibit extracellular signal-regulated kinase 1/2 kinase, phosphatidylinositol 3-kinase, and RhoA kinase also partially suppressed the thrombin-induced IL-6 production, but the effects were smaller than those of the p38 MAP and tyrosine kinase inhibitors. Thus, thrombin induces HGFs to produce IL-6 by activating PAR-1, and the tyrosine kinase- and p38 MAP kinase-dependent pathways, rather than the Ca(2+) signaling pathway, may play a crucial role in the IL-6 production.

Tanaka N ，Morita T ，Nezu A ，Tanimura A ，Mizoguchi I ，Tojyo Y ... -  《JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS》