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

轉化生長因子β（transforming growth factor β；TGFβ）在口腔黏膜下纖維化症（OSF）中扮演了刺激膠原蛋白合成以及減少膠原蛋白分解因而加重纖維化的重要角色。結締組織生長因子（CTGF/CCN2）是轉化生長因子β下游基因產物，幾乎所有轉化生長因子β前纖維化作用和刺激細胞外間質蛋白合成的功能，都需要結締組織生長因子的共同參與。以檳榔鹼（arecoline）刺激人類頰黏膜纖維母細胞（BMFs）可以誘導人類頰黏膜纖維母細胞中結締組織生長因子與環氧酵素二型（cyclooxygenase-2；COX-2）表現。在口腔黏膜下纖維化症的組織中，可以發現結締組織生長因子與環氧酵素二型的過度表現。但是關於口腔黏膜中轉化生長因子β誘導結締組織生長因子表現的訊息傳遞路徑及環氧酵素二型對結締組織生長因子表現的影響仍不清楚。本研究主旨在研究以轉化生長因子β1刺激人類頰黏膜纖維母細胞誘導結締組織生長因子表現的訊息傳導路徑，冀望可以藉由抑制這些訊息傳導路徑成員來達到預防或治療口腔黏膜下纖維化症。我們發現使用TGFβ1 receptor ALK5抑制劑SB431542、Rac1抑制劑NSC23766 、JNK專一性抑制劑 SP600125、p38 MAPK專一性抑制劑SB203580、抗氧化劑N-acetylcysteine （NAC）、泛NOX抑制劑diphenyleneiodonium chloride （DPI）、NOX4抑制劑plumbagin、Src抑制劑（PP2、Src inhibitor-1及 Src si-RNA）做前處理都可以明顯地減少人類頰黏膜纖維母細胞中TGFβ1誘導結締組織生長因子的表現。但ERK專一性抑制劑PD98059、NOX2抑制劑 apocynin 則沒有影響。進一步研究發現，NAC、DPI可以抑制 Src 的活化，但NSC23766 則沒有影響。NAC、DPI 、PP2可以抑制 JNK 的活化，但NSC23766 也沒有影響。NAC、DPI可以抑制 p38 MAPK 的活化，但NSC23766 、PP2沒有影響。NAC、PP2可以抑制 SMAD3 的活化，但NSC23766 、DPI沒有影響。結果顯示，人類頰黏膜纖維母細胞中TGFβ1誘導的結締組織生長因子表現至少經由四條路徑所調控，其分別為TGFβ1/ALK5/Rac1/CTGF(CCN2)、TGFβ1/ALK5/ROS/Src/SMAD3/CTGF、TGFβ1/ALK5/NOX4/ROS/Src/JNK/CTGF 和 TGFβ1/ALK5/NOX4/ROS/p38/CTGF 路徑。茶多酚 EGCG 藉由抑制 JNK 和 p38 MAPK 的磷酸化來阻斷TGFβ1誘導人類頰黏膜纖維母細胞產生的結締組織生長因子蛋白。前列腺素E2 （prostaglandin E2; PGE2）可以抑制人類肺細胞株IMR90中TGFβ1誘導的結締組織生長因子表現，但不能抑制人類頰黏膜纖維母細胞中TGFβ1誘導的結締組織生長因子表現。由於人類頰黏膜纖維母細胞中獨特的訊息傳導路徑致使前列腺素E2無法抑制結締組織生長因子表現以致在口腔黏膜下纖維化症組織中環氧酵素二型能與結締組織生長因子並存。最後發現EGCG亦可以抑制TGFβ1誘導人類頰黏膜纖維母細胞產生的第一型前膠原纖維蛋白表現、減少TGFβ1造成的膠狀膠原收縮、並且可以阻斷 TGFβ1引起的可溶性膠原蛋白的生成。因此茶多酚 EGCG 對於預防以及治療口腔黏膜下纖維化症將有很大的潛力。

關鍵字

結締組織生長因子；茶多酚；口腔黏膜下纖維化症；轉化生長因子β ； CTGF/CCN2

並列摘要

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.

並列關鍵字

CTGF/CCN2 ； Connective tissue growth factor ； EGCG ； Oral submucous fibrosis ； TGFβ1 ； Transforming growth factor β

參考文獻

1. Pindborg, J.J., et al., Oral submucous fibrosis as a precancerous condition. Scand J Dent Res, 1984. 92(3): p. 224-9.

2. Pindborg, J.J. and S.M. Sirsat, Oral submucous fibrosis. Oral Surg Oral Med Oral Pathol, 1966. 22(6): p. 764-79.

3. Angadi, P.V. and K.P. Rekha, Oral submucous fibrosis: a clinicopathologic review of 205 cases in Indians. Oral Maxillofac Surg, 2011. 15(1): p. 15-19.

4. Sirsat, S.M. and J.J. Pindborg, Subepithelial changes in oral submucous fibrosis. Acta Pathol Microbiol Scand, 1967. 70(2): p. 161-73.

5. Yusuf, H. and S.L. Yong, Oral submucous fibrosis in a 12-year-old Bangladeshi boy: a case report and review of literature. Int J Paediatr Dent, 2002. 12(4): p. 271-6.

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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

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