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 Ca²⁺ 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 Ca²⁺ chelator, markedly attenuated the thrombin-induced increase in intracellular Ca²⁺ 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 Ca²⁺ signaling pathway, may play a crucial role in the IL-6 production.