TGFβ1經由 NOX4/Src 訊息傳遞路徑誘導人類頰黏膜纖維母細胞結締組織生長因子表現

Abstract

Transforming growth factor β (TGFβ) has been suggested as the main trigger for the increased collagen production and decreased matrix degradation pathways in oral submucous fibrosis (OSF). Connective tissue growth factor (CTGF/CCN2) is required for most of the increased ECM production and other profibrotic activities generally observed in response to TGFβ. Arecoline induces CCN2 and cyclooxygenase-2 (COX-2) expressions in human buccal mucosal fibroblasts (BMFs). CTGF/CCN2 and COX-2 have been found to overexpress in OSF. However, the molecular mechanisms are not entirely clear. The aims of this study were to investigate the molecular mechanism underlying the TGFβ-induced CTGF/CCN2 expressions in human buccal mucosal fibroblasts (BMFs) and to identify the potential targets for drug intervention or chemoprevention of OSF. Pretreatment with activin receptor-like kinase 5 (ALK5) inhibitor SB431542, Rac1 inhibitor NSC23766, c-Jun NH2-terminal kinase (JNK) inhibitor SP600125, p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580, antioxidant N-acetyl-L-cysteine (NAC), NOX inhibitor diphenylene iodonium (DPI), NOX4 inhibitor plumbagin, and Src inhibitors (PP2, Src inhibitor-1, and Src si-RNA) significantly reduced TGFβ1-induced CTGF/CCN2 synthesis in BMFs, but not extracellular signal-regulated kinase (ERK) inhibitor PD98059, or NOX2 inhibitor apocynin. Furthermore, the phosphorylations of Src were inhibited by NAC and DPI but not NSC23766. The phosphorylations of JNK were inhibited by NAC, DPI, and PP2 but not NSC23766. The phosphorylations of p38 MAPK were inhibited by NAC and DPI but not NSC23766 or PP2. The phosphorylations of SMAD3 were inhibited by NAC and PP2 but not NSC23766 or DPI. These results revealed that multiple parallel signal transduction pathways together mediated the TGFβ1-induced CTGF/CCN2 expression in BMFs. At least four major pathways TGFβ1/ALK5/Rac1/CTGF(CCN2), TGFβ1/ALK5/ROS/Src/SMAD3/CTGF, TGFβ1/ALK5/NOX4/ROS/Src/JNK/CTGF, and TGFβ1/ALK5/NOX4/ROS/p38/CTGF were identified. Epigallocatechin-3-gallate (EGCG) blocked TGFβ1-induced CTGF/CCN2 synthesis by inhibiting the phosphorylation of Src, JNK, and p38 MAPK. Prostaglandin E2 (PGE2) inhibited the TGFβ1-induced CTGF/CCN2 synthesis in human fetal lung fibroblasts IMR90 but not in BMFs. The exceptional signal transduction pathways of TGFβ1-induced CTGF/CCN2 production in BMFs contribute to the resistance of PGE2 downregulation of CTGF/CCN2 expression; therefore, the CTGF/CCN2 levels are maintained in the OSF tissues in the presence of COX-2. In addition, EGCG inhibits the expression of TGFβ1-induced type I procollagen, reduces the excessive soluble collagens produced by TGFβ1-treated BMFs, and attenuates the fibroblast-mediated gel contraction stimulated by TGFβ1. Therefore, EGCG may serve as a useful agent in controlling OSF.