Graduate Institute of Clinical Medicine, College of Medicine, Taipei Medical University, Taiwan, China.
Acta Pharmacol Sin. 2012 Jan;33(1):49-56. doi: 10.1038/aps.2011.178.
To investigate the signaling pathways involved in thrombin-induced connective tissue growth factor (CTGF) expression in rat vascular smooth muscle cells (VSMCs).
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.
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.
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.
研究凝血酶诱导大鼠血管平滑肌细胞（VSMCs）结缔组织生长因子（CTGF）表达涉及的信号通路。
在原代大鼠主动脉平滑肌细胞（RASMCs）和大鼠 VSMC 系（A10）上进行实验。使用 Western blot 法测定 CTGF 蛋白水平。用荧光素酶报告基因和显性负突变体（DN）研究介导凝血酶诱导 CTGF 表达的信号通路。
凝血酶（0.3-3.0 U/mL）引起 RASMCs 和 A10 细胞中 CTGF 表达的浓度和时间依赖性增加。用蛋白酶激活受体 1（PAR-1）拮抗剂 SCH79797（0.1 μmol/L）预处理 A10 细胞可显著阻断凝血酶诱导的 CTGF 表达，而 PAR-4 拮抗剂 tcY-NH2（30 μmol/L）则没有效果。PAR-1 激动剂 SFLLRN-NH2（300 μmol/L）诱导 CTGF 表达，而 PAR-4 激动剂 GYPGQV-NH2（300 μmol/L）则没有效果。凝血酶（1 U/mL）引起 c-Jun N-末端激酶（JNK）的时间依赖性磷酸化。用 JNK 抑制剂 SP600125（3-30 μmol/L）预处理或转染 JNK1/2 的 DN 可显著减弱凝血酶诱导的 CTGF 表达。凝血酶（0.3-3.0 U/mL）增加激活蛋白-1（AP-1）-荧光素酶活性，该活性可被 JNK 抑制剂 SP600125 抑制。AP-1 抑制剂姜黄素（1-10 μmol/L）浓度依赖性地减弱凝血酶诱导的 CTGF 表达。
凝血酶作用于 PAR-1 以激活 JNK 信号通路，进而启动 AP-1 激活，最终诱导 VSMCs 中的 CTGF 表达。
