PI3K/Akt 通路介导 TGF-β2 诱导的人视网膜色素上皮细胞 I 型胶原的表达。
The PI3K/Akt pathway mediates the expression of type I collagen induced by TGF-β2 in human retinal pigment epithelial cells.
机构信息
Department of Ophthalmology, Faculty of Medicine, Oita University, Hasama-machi, Yufu-shi, Oita 879-5593, Japan.
出版信息
Graefes Arch Clin Exp Ophthalmol. 2012 Jan;250(1):15-23. doi: 10.1007/s00417-011-1766-x. Epub 2011 Aug 20.
PURPOSE
Transforming growth factor (TGF)-β is a key mediator of proliferative vitreoretinopathy, but the cellular mechanisms by which TGF-β induces extracellular matrix protein (ECM) synthesis are not fully understood. This study examined whether the PI3K/Akt pathway is involved in TGF-β2-induced collagen expression in human retinal pigment epithelial cells.
METHODS
Human retinal pigment epithelial cells ARPE-19 were cultured and stimulated with TGF-β2. The role of the PI3K/Akt pathway was evaluated using the biochemical inhibitor, wortmannin. The effect of wortmannin on the expression of type I collagen mRNA (COL1A1, COL1A2) induced by TGF-β2 was evaluated by real-time RT-PCR. The effect of wortmannin on the synthesis of type I collagen induced by TGF-β2 was assessed by an immunocytochemical analysis with anti-type I collagen antibody. Luciferase reporter assays were performed to examine the effect of wortmannin on the transcriptional activities of COL1A2. A luciferase assay using a mutation construct of the Smad binding site in COL1A2 promoter (Smad-mut/Luc) was also performed to examine the crosstalk between the Smad pathway and the PI3K/Akt pathway. The effects of wortmannin on the transcriptional activity of Smad3 were also examined using CAGA12-Luc. Moreover, the effect of wortmannin on TGF-β2-induced Smad7 mRNA expression was evaluated.
RESULTS
The biochemical blockade of PI3K/Akt activation inhibited TGF-β2-induced type I collagen mRNA expression and type I collagen synthesis. The blockade of PI3K/Akt pathway inhibited the increase in COL1A2 promoter activities when induced by TGF-β2 and reduced TGF-β2 induction of Smad-mut/Luc promoter activity and CAGA12-Luc activity. Moreover, wortmannin increased the TGF-β2-induced Smad7 mRNA expression levels.
CONCLUSIONS
The PI3K/Akt pathway plays a role in relaying the TGF-β2 signal to induce type I collagen synthesis in the retinal pigment epithelium through Smad-dependent and Smad-independent pathways.
目的
转化生长因子-β（TGF-β）是增生性玻璃体视网膜病变的关键介质，但TGF-β诱导细胞外基质蛋白（ECM）合成的细胞机制尚不完全清楚。本研究探讨了 PI3K/Akt 通路是否参与了 TGF-β2 诱导人视网膜色素上皮细胞胶原表达。
方法
培养人视网膜色素上皮细胞 ARPE-19 并刺激其表达 TGF-β2。采用 PI3K/Akt 通路生化抑制剂渥曼青霉素（wortmannin）评估其作用。通过实时 RT-PCR 检测 wortmannin 对 TGF-β2 诱导的Ⅰ型胶原 mRNA（COL1A1、COL1A2）表达的影响。通过用抗Ⅰ型胶原抗体进行免疫细胞化学分析评估 wortmannin 对 TGF-β2 诱导的Ⅰ型胶原合成的影响。进行荧光素酶报告基因实验以检测 wortmannin 对 COL1A2 转录活性的影响。还进行了 COL1A2 启动子中 Smad 结合位点突变构建体（Smad-mut/Luc）的荧光素酶测定，以研究 Smad 通路与 PI3K/Akt 通路之间的串扰。还使用 CAGA12-Luc 检测了 wortmannin 对 Smad3 转录活性的影响。此外，还评估了 wortmannin 对 TGF-β2 诱导的 Smad7 mRNA 表达的影响。
结果
PI3K/Akt 激活的生化阻断抑制了 TGF-β2 诱导的Ⅰ型胶原 mRNA 表达和Ⅰ型胶原合成。PI3K/Akt 通路阻断抑制了 TGF-β2 诱导的 COL1A2 启动子活性增加，并降低了 TGF-β2 诱导的 Smad-mut/Luc 启动子活性和 CAGA12-Luc 活性。此外，wortmannin 增加了 TGF-β2 诱导的 Smad7 mRNA 表达水平。
结论
PI3K/Akt 通路通过 Smad 依赖性和 Smad 非依赖性途径在视网膜色素上皮细胞中发挥作用，将 TGF-β2 信号转导至诱导Ⅰ型胶原合成。
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