Thrombin and PAR-1-AP increase proinflammatory cytokine expression in C6 cells

Background. In addition to a recognized role in the coagulation cascade, thrombin is known to have other functions via G protein-coupled receptors, including protease-activated receptor-1 (PAR-1). To investigate the relationship between PAR-1 activation and proinflammatory cytokine expression, we studied the responsiveness of C6 cells to thrombin and to the agonist PAP-1-activating peptide (PAR-1-AP). Materials and methods. Cultured C6 rat glioma cells were stimulated with human a-thrombin or PAR-1-AP. To study mRNA expression changes, total RNA was isolated from the C6 cells, reverse transcribed, and amplified by real-time polymerase chain reaction. Three proinflammatory cytokines were studied: interleukin-6 (IL-6), interleukin-1 beta (IL-1 beta), and tumor necrosis factor-alpha (TNF-alpha). To measure cytokine release, cell-free supernatants were assayed using enzyme-linked immunosorbent assay (ELISA). Results. By quantitative real time reverse transcriptase polymerase chain reaction, thrombin (5 U/mL) exposure significantly increased mRNA expression of the proinflammatory cytokines: IL-6 (2.8 +/- 0.4, multiple of control), IL-1 beta (4.8 +/- 1.6), and TNF-alpha (16.5 +/- 4.2). Effects on IL-6 mRNA expression were dose-dependent and matched by increments in IL-6 protein secretion. Effects of thrombin on IL-6 mRNA expression could be inhibited by hirudin. PAR-1-AP exposure also significantly increased mRNA expression of IL-6, IL-1 beta and TNF-alpha. PAR-1 mRNA is expressed in C6 cells. Conclusion. Both thrombin and its agonist, PAR-1-AP, significantly increased mRNA expression of proinflammatory cytokines in C6 glioma cells via PAR-1 activation. (c) 2005 Elsevier Inc. All rights reserved.