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* Graduate Institute of Medical Sciences,
Department of Pharmacology,
Department of Microbiology and Immunology,
School of Respiratory Therapy, College of Medicine, Taipei Medical University, Taipei, Taiwan;
¶ Angiogenesis Research Center, Laboratory of Molecular and Cellular Toxicology, Institute of Toxicology, College of Medicine, National Taiwan University, Taipei, Taiwan;
|| Department of Thoracic and Cardiovascular Surgery and
# Department of Pulmonary Medicine, Taipei Medical University-Municipal Wangfang Hospital, Taipei, Taiwan; and
** Taipei Medical University-Shuang Ho Hospital, Taipei county, Taiwan
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 investigated the role of apoptosis signal-regulating kinase 1 (ASK1) 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-induced CTGF expression and CTGF-luciferase activity were inhibited by a protease-activated receptor 1 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 induced increases in JNK phosphorylation and kinase activity, which were attenuated by ASK1DN. Furthermore, SCH79797 diminished the thrombin-induced ASK1 and JNK 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 construct. 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 ASK1DN, JNK1/2DN, and SP600125. These results suggest for the first time that thrombin, acting through protease-activated receptor 1, activates the ASK1/JNK signaling pathway, which in turn initiates c-Jun/AP-1 activation and recruitment of c-Jun to the CTGF promoter and ultimately induces CTGF expression in human lung fibroblasts.
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1 This work was supported by grants (NSC96-2320-B-038-029-MY3 and NSC97-2320-B-038-014-MY3) from the National Science Council, Taiwan.
2 Address correspondence and reprint requests to Dr. Bing-Chang Chen, School of Respiratory Therapy and Dr. Chien-Huang Lin, Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan, 250 Wu-Hsing Street, Taipei 110, Taiwan. E-mail addresses: bcchen{at}tmu.edu.tw and chlin{at}tmu.edu.tw
3 Abbreviations used in this paper: PAR, protease-activated receptor; CTGF, connective tissue growth factor; BCE-1, basal control element 1; Sp1, specificity protein 1; ASK1, apoptosis signal-regulating kinase 1; ActD, actinomycin D; CHX, cycloheximide; DN, dominant- negative mutant; NHALF, normal human adult lung fibroblast; MBP, myelin basic protein; NEAA, nonessential amino acid; ChIP, chromatin immunoprecipitation; MKK, MAPK kinase; MKKK, MAPK kinase kinase.
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