Molecular Mechanisms for Thrombin-stimulated Connective Tissue Growth Factor Production in Human Gingival Fibroblasts

• Main Authors: Wei-Ni Lin, 林威妮
• Other Authors: 郭彥彬
• Format: Others
• Language: zh-TW
• Published: 2011
• Online Access: http://ndltd.ncl.edu.tw/handle/13245799338336065332

碩士 === 國立臺灣大學 === 臨床牙醫學研究所 === 99 === Connective tissue growth factor (CTGF) is associated with the onset and progression of fibrosis in many human tissues and was found to overexpress in the tissue of gingival overgrowth. It has been reported that, in spite of a proper recall program after surgical treatment, the recurrent rate of gingival overgrowth in 18 months was still up to 34%. Thrombin has been implicated in lung fibrosis, with its capacity of inducing CTGF expression in lung fibroblasts. So we postulate that, the recruitment of platelets and normal clotting factors, like thrombin, early in the wound healing process could be responsible for the rapid re-growth of fibrotic gingival tissue. In this study, we showed that thrombin caused a concentration- and time-dependent increase in CTGF expression in human gingival fibroblasts (GFs). The effect of thrombin could be mimicked with the protease-activated receptor 1 (PAR1) agonist peptide, SFLLRN, and could be completely inhibited by a serine protease inhibitor, PPACK, indicating that thrombin mediated this effect via the proteolytic cleavage and activation of PAR1. We further used several inhibitors of mitogen-activated protein kinase (MAPK) pathway and reactive oxygen species (ROS) pathway, and a popular chemopreventive agent, EGCG, to investigate the key signaling molecules in thrombin-induced CTGF expression in GFs. The results of western blotting revealed that, the inhibitors of MAPK pathway, ASK1 inhibitor (thioredoxin), JNK inhibitor (SP600125), but not PI3K inhibitor (LY294002), ERK inhibitor (PD98059), p38 MAPK inhibitor (SB203580), could significantly reduce the level of thrombin-induced CTGF. In addition, antioxidant N-acetyl-L-cysteine (NAC), Rac-GTPase inhibitor (NSC-23766), NADPH oxidase inhibitors (apocynin and DPI), but not 5-lipoxygenase activating protein (FLAP) inhibitor (MK886), had the inhibitory effects on the thrombin-induced CTGF. Furthermore, AP-1 inhibitor (curcumin) and EGCG could also attenuate the stimulatory effect of thrombin on CTGF expression with a dose-dependent manner. These results suggested that thrombin-induced CTGF expression in GFs could be mediated by PAR1, ROS, ASK1, JNK and AP-1 pathways, and the ROS-generating NADPH oxidase may play a part in the pathways as well. EGCG also displays a chemopreventive potential in treating or preventing the recurrence of gingival overgrowth.