Thrombin-induced connective tissue growth factor expression in human lung fibroblasts requires ASK1/JNK/AP-1 pathway

Chien-Huang Lin1, Chung-Chi Yu1, Ming-Jen Hsu1, Min-Liang Kuo2, Bing-Chang Chen1
1Taipei Medical University, Taipei, Taiwan, 2National Taiwan University, Taipei, Taiwan

In this study, we investigated the role of apoptosis signal-regulating kinase 1 (ASK1) in thrombin-induced AP-1 activation and connective tissue growth factor (CTGF) expression in human lung fibroblasts. The thrombin-mediated CTGF expression and CTGF-luciferase activity were inhibited by the dominant-negative mutants (DNs) of ASK1, JNK1, JNK2, an AP-1 inhibitor (curcumin), and a PAR1 receptor antagonist (SCH 79797), but not by a PAR4 antagonist (tcY-NH2). Thrombin caused ASK1 Ser967 dephosphorylation, dissociation of ASK1 and 14-3-3, and a subsequent increase in ASK1 activity. Thrombin-induced JNK activation was inhibited by ASK1DN. Thrombin caused increases in c-Jun phosphorylation, the formation of an AP-1-specific DNA-protein complex, and the recruitment of c-Jun to response elements on the CTGF promoter. The thrombin-induced CTGF-luciferase activity was major controlled by the -747 to -184 bp upstream of transcription start site on human CTGF promoter. Furthermore, the thrombin-induced CTGF-luciferase activity was attenuated by transfection with the deleted AP-1 binding site construct. Furthermore, thrombin-mediated AP-1 activation was inhibited by ASK1DN and SP600125. These results suggest for the first time that thrombin, acting through PAR1, activates the ASK1/JNK signaling pathway, which in turn initiates c-Jun/AP-1 activation and recruitment to CTGF promoter, and finally induces CTGF expression in human lung fibroblasts.