Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei 11031, Taiwan.
Graduate Institute of Toxicology, College of Medicine, National Taiwan University, Taipei 100, Taiwan; Graduate Institute of Biomedical Sciences, College of Life Science, National Taiwan University, Taipei 106, Taiwan.
Biochem Pharmacol. 2014 Apr 1;88(3):402-11. doi: 10.1016/j.bcp.2014.01.030. Epub 2014 Jan 31.
Endothelin-1 (ET-1) acts as a key mediator of vasoconstriction and tissue repair. Overproduction of connective tissue growth factor (CTGF) underlies the development of lung fibrosis. ET-1 induces expression of matrix-associated genes in lung fibroblasts, however, little is known about the signaling pathway of CTGF expression caused by ET-1. In this study, we found that ET-1 caused concentration- and time-dependently increases in CTGF expression in human embryonic lung fibroblast cell line (WI-38). ET-1-induced CTGF expression was inhibited by BQ123 (ETAR antagonist), but not BQ788 (ETBR antagonist). Moreover, ET-1-induced CTGF expression was significantly reduced by JNK inhibitor (SP600125), the dominant-negative mutants of JNK1/2 (JNK1/2 DN), and AP-1 inhibitor (curcumin). ET-1 induced phosphorylations of JNK and c-Jun in time-dependent manners. AP-1 luciferase activity was concentration-dependently increased by ET-1, and this effect was attenuated by SP600125. We also found that ET-1-induced CTGF expression was most controlled by the AP-1 binding region of CTGF promoter. ET-1-indiced CTGF luciferase activity was predominately controlled by the sequence -747 to -408 bp upstream of the transcription start site on the human CTGF promoter. Furthermore, ET-1 caused the formation of AP-1-specific DNA-protein complex and the recruitment of c-Jun to the CTGF promoter. Moreover, we found that ET-1 induced α-smooth muscle actin (α-SMA) expression, which was inhibited by BQ123, SP600125, curcumin, and anti-CTGF antibody. These results suggest that ET-1 stimulates expressions of CTGF and α-SMA through ETAR/JNK/AP-1 signaling pathway, and CTGF is required for ET-1-induced α-SMA expression in human lung fibroblasts.
内皮素-1（ET-1）作为血管收缩和组织修复的关键介质发挥作用。结缔组织生长因子（CTGF）的过度产生是肺纤维化发展的基础。ET-1 诱导肺成纤维细胞中基质相关基因的表达，但对于 ET-1 引起的 CTGF 表达的信号通路知之甚少。在这项研究中，我们发现 ET-1 导致人胚肺成纤维细胞系（WI-38）中 CTGF 表达呈浓度和时间依赖性增加。ETAR 拮抗剂 BQ123 但不是 ETBR 拮抗剂 BQ788 抑制 ET-1 诱导的 CTGF 表达。此外，JNK 抑制剂（SP600125）、JNK1/2 的显性失活突变体（JNK1/2 DN）和 AP-1 抑制剂（姜黄素）显著降低 ET-1 诱导的 CTGF 表达。ET-1 以时间依赖性方式诱导 JNK 和 c-Jun 的磷酸化。AP-1 荧光素酶活性被 ET-1 浓度依赖性地增加，而 SP600125 则减弱了这种作用。我们还发现，ET-1 诱导的 CTGF 表达主要受 CTGF 启动子中 AP-1 结合区的控制。ET-1 诱导的 CTGF 荧光素酶活性主要受人类 CTGF 启动子转录起始位点上游 -747 至-408bp 序列的控制。此外，ET-1 引起 AP-1 特异性 DNA-蛋白质复合物的形成和 c-Jun 向 CTGF 启动子的募集。此外，我们发现 ET-1 诱导α-平滑肌肌动蛋白（α-SMA）表达，该表达被 BQ123、SP600125、姜黄素和抗 CTGF 抗体抑制。这些结果表明，ET-1 通过 ETAR/JNK/AP-1 信号通路刺激 CTGF 和 α-SMA 的表达，并且 CTGF 是 ET-1 诱导人肺成纤维细胞中α-SMA 表达所必需的。
