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).


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.


CONCLUSIONS
Both thrombin and its agonist, PAR-1-AP, significantly increased mRNA expression of pro-inflammatory cytokines in C6 glioma cells via PAR-1 activation.
Fan
Yongyi
Y
Department of Surgery, University of Michigan, Ann Arbor, Michigan 48109, USA.
Zhang
Weizhen
W
Mulholland
Michael
M
D000818:Animals; D001932:Brain Neoplasms; D045744:Cell Line, Tumor; D016207:Cytokines / Q000235:genetics*; D005343:Fibrinolytic Agents / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects / Q000276:immunology; D005910:Glioma; D006490:Hemostatics / Q000494:pharmacology*; D006629:Hirudins / Q000494:pharmacology; D007375:Interleukin-1 / Q000235:genetics; D015850:Interleukin-6 / Q000235:genetics; D009842:Oligopeptides / Q000494:pharmacology*; D000073718:Pancreatitis-Associated Proteins; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D013917:Thrombin / Q000494:pharmacology*; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics
D016207:Cytokines; D005343:Fibrinolytic Agents; D006490:Hemostatics; D006629:Hirudins; D007375:Interleukin-1; D015850:Interleukin-6; D009842:Oligopeptides; C462999:PAR-1-activating peptide; D000073718:Pancreatitis-Associated Proteins; C000612141:REG3A protein, human; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D013917:Thrombin