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


CONCLUSIONS
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.
Graduate Institute of Clinical Medicine, College of Medicine, Taipei Medical University, Taiwan, China.
Wen-chin
WC
Ko
Bing-chang
BC
Chen
Ming-jen
MJ
Hsu
Chia-ti
CT
Tsai
Chuang-ye
CY
Hong
Chien-huang
CH
Lin
D000818:Animals; D002460:Cell Line; D055513:Connective Tissue Growth Factor / Q000235:genetics / Q000378:metabolism*; D006490:Hemostatics / Q000494:pharmacology; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D020935:MAP Kinase Signaling System / Q000187:drug effects* / Q000502:physiology; D009131:Muscle, Smooth, Vascular / Q000166:cytology; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000187:drug effects* / Q000502:physiology; D051381:Rats; D044463:Receptor, PAR-1 / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D018179:Receptors, Thrombin / Q000819:agonists / Q000037:antagonists & inhibitors; D013917:Thrombin / Q000494:pharmacology*; D018808:Transcription Factor AP-1 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D010975:Platelet Aggregation Inhibitors
D006490:Hemostatics; D044463:Receptor, PAR-1; D018179:Receptors, Thrombin; D018808:Transcription Factor AP-1; D055513:Connective Tissue Growth Factor; D048031:JNK Mitogen-Activated Protein Kinases; D013917:Thrombin; C112908:protease-activated receptor 4; D010975:Platelet Aggregation Inhibitors