Studies on the Molecular Mechanism of Thrombin-Induced Connective Tissue Growth Factor (CTGF) Expression in Lung Fibroblasts

Thrombin, a serine protease, is a well-known coagulation factor generated in vascular injury and also plays an important role in lung inflammatory diseases. The levels of thrombin are increased in bronchoalveolar lavage fluid from patients with acute respiratory distress syndrome, pneumonia, and asthma, as well as in animal models of fibrotic lung disease. Several reports indicated that thrombin can induce the production of fibrotic mediators, such as transforming growth factor-β (TGF-β), fibronectin, and collagen in lung fibroblasts. Connective tissue growth factor (CTGF or CCN2) is a member of the CCN family which is an immediate early gene. Furthermore, several studies have shown that CTGF overproduction underlies the development of lung fibrosis. Previous studies have shown that thrombin is a potent inducer of CTGF production in lung fibroblast. However, little information is known about the signaling pathway of CTGF expression caused by thrombin. Our preliminary studies indicate that activation of apoptosis signal-regulating kinase 1 (ASK1) is involved in CTGF expression caused by thrombin in human lung fibroblasts. We also found that thrombin-induced CTGF expression was most controlled by the region between -747 to -184 base pairs of CTGF promoter. It has been identified that the region of -747 to -184 exists the consensus sequences of the transcription factors, AP-1 and STAT. In this study, we will investigate the signaling cascade and the requirement of AP-1 and/or STAT participating in thrombin-mediated CTGF expression. Moreover, we found that thrombin increased the half-life of CTGF mRNA, suggesting thrombin-mediated CTGF expression maybe partly via regulation of CTGF mRNA stability. The Central Hypothesis of this proposal is that thrombin induces ASK1/JNK and c-Src/JAK signaling cascade resulting in AP-1 and 1 2 STAT activation, and causes MK-2/HuR activation to stabilize CTGF mRNA, which in turns induces CTGF expression in human lung fibroblasts. The overall objective of this project is to elucidate the molecular mechanism of thrombin-induced CTGF expression so that effective interventions can be developed to prevent lung fibrosis. The hypotheses and specific aims are described below: Specific Aim 1 (1st ~2nd year): To investigate the roles of ASK1 and JAK signaling cascade in thrombin-induced CTGF expression in lung fibroblasts. Hypothesis 1: Thrombin-induced CTGF expression in fibroblasts is mediated by ASK1 and JAK signaling cascade Specific Aim 2 (2nd year): To investigate the requirement of AP-1 and/or STAT on thrombin-induced CTGF expression in fibroblasts. Hypothesis 2: The activation and interaction of transcription factors, AP-1 and/or STAT, are required for CTGF expression by thrombin in fibroblasts. Specific Aim 3 (3rd year): To characterize the post-transcriptional mechanism involved in CTGF mRNA stability caused by thrombin in lung fibroblasts. Hypothesis 3: Activation of the RNA binding protein, HuR, is required for CTGF mRNA stability and consequently CTGF protein expression caused by thrombin