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 alpha-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-1beta (IL-1beta), 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-1beta (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-1beta 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 pro-inflammatory cytokines in C6 glioma cells via PAR-1 activation.
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 ratglioma cells were stimulated with human alpha-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-1beta (IL-1beta), 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-1beta (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-1beta 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 pro-inflammatory cytokines in C6 glioma cells via PAR-1 activation.
Authors: Akanksha Gupta; Bruce Gerlitz; Mark A Richardson; Christopher Bull; David T Berg; Samreen Syed; Elizabeth J Galbreath; Barbara A Swanson; Bryan E Jones; Brian W Grinnell Journal: J Am Soc Nephrol Date: 2008-12-17 Impact factor: 10.121
Authors: Christoph Anthoni; Janice Russell; Katherine C Wood; Karen Y Stokes; Thorsten Vowinkel; Daniel Kirchhofer; D Neil Granger Journal: J Exp Med Date: 2007-06-11 Impact factor: 14.307
Authors: Jenell R Smith; Peter P Syre; Shaina A Oake; Kristen J Nicholson; Christine L Weisshaar; Katrina Cruz; Robert Bucki; Bethany C Baumann; Paul A Janmey; Beth A Winkelstein Journal: PLoS One Date: 2013-11-20 Impact factor: 3.240