Thrombin induced connective tissue growth factor expression in rat vascular smooth muscle cells via the PAR-1/JNK/AP-1 pathway

Aim:To investigate the signaling pathways involved in thrombin-induced connective tissue growth factor (CTGF) expression in rat vascular smooth muscle cells (VSMCs).Methods:Experiments were preformed on primary rat aortic smooth muscle cells (RASMCs) and a rat VSMC line (A10). CTGF protein levels were measured using Western blotting. Luciferase reporter genes and dominant negative mutants (DNs) were used to investigate the signaling pathways mediating the induction of CTGF expression by thrombin.Results:Thrombin (0.3-3.0 U/mL) caused a concentration-and time-dependent increase in CTGF expression in both RASMCs and A10 cells. Pretreating A10 cells with the protease-activated receptor 1 (PAR-1) antagonist SCH79797 (0.1 μmol/L) significantly blocked thrombin-induced CTGF expression, while the PAR-4 antagonist tcY-NH 2 (30 μmol/L) had no effect. The PAR-1 agonist SFLLRN-NH ₂ (300 μmol/L) induced CTGF expression, while the PAR-4 agonist GYPGQV-NH ₂ (300 μmol/L) had no effect. Thrombin (1 U/mL) caused time-dependent phosphorylation of c-Jun N-terminal kinase (JNK). Pretreating with the JNK inhibitor SP600125 (3-30 μmol/L) or transfection with DNs of JNK1/2 significantly attenuated thrombin-induced CTGF expression. Thrombin (0.3-3.0 U/mL) increased activator protein-1 (AP-1)-luciferase activity, which was inhibited by the JNK inhibitor SP600125. The AP-1 inhibitor curcumin (1-10 μmol/L) concentration-dependently attenuated thrombin-induced CTGF expression.Conclusion:Thrombin acts on PAR-1 to activate the JNK signaling pathway, which in turn initiates AP-1 activation and ultimately induces CTGF expression in VSMCs.