| Summary: | 博士 === 臺北醫學大學 === 醫學科學研究所 === 97 === Thrombin plays an important role in lung inflammatory diseases. Thrombin can induce connective tissue growth factor (CTGF) expression in lung fibroblasts. However, little is known about the signaling pathway in thrombin-induced CTGF expression. In this study, we first investigated the roles of apoptosis signal-regulating kinase 1 (ASK1), JNK, and AP-1 in thrombin-induced CTGF expression in human lung fibroblasts. Thrombin caused a concentration- and time-dependent increase in CTGF expression in WI-38 cells and primary lung fibroblasts. Thrombin also induced CTGF mRNA and CTGF-luciferase activity in WI-38 cells. Thrombin-induced CTGF expression and CTGF-luciferase activity were inhibited by a PAR1 antagonist (SCH79797), the dominant-negative mutants (DNs) of ASK1 and JNK1/2, and an AP-1 inhibitor (curcumin). Thrombin caused ASK1 Ser967 dephosphorylation, the dissociation of ASK1 and 14-3-3, and a subsequent increase in ASK1 activity. Thrombin also induced increases in both JNK phosphorylation and kinase activity, which were attenuated by ASK1DN. Furthermore, SCH79797 diminished the thrombin-induced ASK1 and JNK kinase activities. Thrombin-induced CTGF-luciferase activity was predominately controlled by the sequence -747 to -184 bp upstream of the transcription start site of the human CTGF promoter and was attenuated by transfection with the deleted AP-1 binding site constructs. Thrombin caused increases in c-Jun phosphorylation, the formation of an AP-1-specific DNA-protein complex, and the recruitment of c-Jun to the CTGF promoter. Furthermore, thrombin-mediated AP-1 activation was inhibited by SCH79797, ASK1DN, JNK1/2DN, and SP600125. These results indicate that in human lung fibroblasts thrombin might activate PAR1/ASK1/JNK/c-Jun/AP-1 pathway, which in turn initiates c-Jun/AP-1 activation, and finally induced CTGF protein expression
We further to explore the role of Ras, Raf-1, ERK, IKKa/b, and transcription factor NF-?羠 in thrombin-induced CTGF expression in WI-38 cells. The thrombin-induced increase in CTGF expression was inhibited by a MEK inhibitor (PD98059) but not a p38MAPK inhibitor (SB203580). Furthermore, the CTGf expression was inhibited by a Ras inhibitor (manumycin A), a Raf inhibitor (GW5074), an IkB?? phosphorylation inhibitor (Bay117082), and the DN of IkB (IkBM). Thrombin-induced increases in CTGF-luciferase activity also inhibited by manumycin A, GW5074, PD98059, and Bay117082. Moreover, thrombin induced increases in Ras activity, Raf-1, and IKKa/b phosphorylation, and the Raf-1 phosphorylation was attenuated by manumycin A. Thrombin-induced ERK phosphorylation was also inhibited by manumycin A and GW5074. Furthermore, thrombin-induced c-Jun phosphorylation was inhibited by PD98059. These results indicate that in lung fibroblasts, thrombin might activate the Ras/Raf-1/ERK/c-Jun and IKKa/b/NF-kB signaling pathway, which in turn cause CTGF expression.
Taken together, our results suggest for the first time that thrombin, acting through PAR1, activates the ASK1/JNK/c-Jun/AP-1, Ras/Raf/ERK/c-Jun, and IKKa/b/NF-kB signaling pathway, which in turn initiates activation of the CTGF promoter, and ultimately induces CTGF expression in human lung fibroblasts.
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