Objectives: It has been shown that activation of the protease-activated receptor-1 (PAR-1) by thrombin induces interleukin-6 (IL-6) production in human gingival fibroblasts (HGFs). We investigated the signaling mechanism for the PAR-1-mediated IL-6 production. Method: Changes in cytosolic Ca²⁺ concentration ([Ca²⁺]i) were studied with an ARGUS HiSCA imaging system in cultured HGFs loaded with fura-2. Expression of mRNA for PARs and IL-6 was analyzed by RT-PCR. IL-6 production was determined with ELISA. Result: a-Thrombin caused a transient increase in [Ca²⁺]i in HGFs, and the maximum response was obtained with 10 nM a-thrombin. This Ca²⁺ response was mimicked by the PAR-1 agonist peptide SFLLRN. The PCR analysis showed that HGFs express PAR-1 mRNA. Stimulation with a-TB or SFLLRN induced IL-6 proiduction and expression of IL-6 mRNA To assess whether the Ca²⁺ signaling is involved in IL-6 production, HGFs were loaded with the intracellular Ca²⁺ chelator BAPTA. The loading with BAPTA markedly attenuated the thrombin-induced increase in [Ca²⁺]i, but the IL-6 production was not inhibited by the same treatment. In contrast, the tyrosine kinase inhibitors, genistein and tyrphostin 23, strongly suppressed the thrombin-induced IL-6 production. The p38 MAP kinase inhibitor SB203580 also inhibited the IL-6 production. Conclusion: The present study indicates that the Ca²⁺ signaling pathway does not play an essential role in the IL-6 production in HGFs. PAR-1 may mediate IL-6 production through tyrosine kinase and MAP kinase-dependent pathway.

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