Kimoto Kenichi, Nakatsuka Kazuo, Matsuo Noritaka, Yoshioka Hidekatsu
Department of Ophthalmology, Faculty of Medicine, Oita University, Hasama-machi, Japan.
Invest Ophthalmol Vis Sci. 2004 Jul;45(7):2431-7. doi: 10.1167/iovs.03-1276.
Transforming growth factor (TGF)-beta has been implicated as the key mediator of proliferative vitreoretinopathy, but the cellular mechanisms by which TGF-beta induces extracellular matrix protein (ECM) synthesis are not fully understood. The current study was conducted to examine whether the mitogen-activated protein kinase (MAPK) pathway is involved in TGF-beta 2-induced collagen expression in retinal pigment epithelial cells.
Human retinal pigment epithelial cells ARPE-19 were cultured and stimulated with various concentrations of TGF-beta 2. The type I collagen gene (COL1A1, COL1A2) expression induced by TGF-beta 2 was evaluated by real-time RT-PCR. Synthesis of type I collagen was evaluated by the concentration of the C-terminal propeptide of type I (PICP) in the medium. The activation of MAPK pathways by TGF-beta 2 was assessed by immunoblot with anti-phospho-p38 and anti-phospho-extracellular signal-regulated kinase (ERK)1/2 antibodies. The role of MAPK was assessed using biochemical inhibitors. To examine the transcriptional activities of COL1A1 and COL1A2, luciferase reporter assays were also performed.
mRNA expression of COL1A1 and COL1A2 and type I collagen synthesis were activated by TGF-beta 2. Both ERK and p38 MAPK pathways were also activated by TGF-beta 2. The biochemical blockade of p38 MAPK activation, but not ERK activation, inhibited TGF-beta 2-induced type I collagen mRNA expression and type I collagen synthesis. Moreover, blockade of the p38 MAPK pathway inhibited the increase in both COL1A1 and COL1A2 promoter activities when induced by TGF-beta 2.
TGF-beta 2 activates p38 MAPK and p38 MAPK plays a role in relaying the TGF-beta 2 signal to type I collagen production in the retinal pigment epithelium.
转化生长因子（TGF）-β被认为是增殖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）合成的细胞机制尚未完全明确。本研究旨在探讨丝裂原活化蛋白激酶（MAPK）途径是否参与TGF-β2诱导的视网膜色素上皮细胞中胶原蛋白的表达。
培养人视网膜色素上皮细胞ARPE-19，并用不同浓度的TGF-β2进行刺激。通过实时逆转录聚合酶链反应（RT-PCR）评估TGF-β2诱导的I型胶原蛋白基因（COL1A1、COL1A2）表达。通过培养基中I型胶原蛋白C末端前肽（PICP）的浓度评估I型胶原蛋白的合成。用抗磷酸化p38和抗磷酸化细胞外信号调节激酶（ERK）1/2抗体进行免疫印迹，评估TGF-β2对MAPK途径的激活作用。使用生化抑制剂评估MAPK的作用。为检测COL1A1和COL1A2的转录活性，还进行了荧光素酶报告基因分析。
TGF-β2激活了COL1A1和COL1A2的mRNA表达以及I型胶原蛋白的合成。ERK和p38 MAPK途径也被TGF-β2激活。p38 MAPK激活的生化阻断而非ERK激活的阻断，抑制了TGF-β2诱导的I型胶原蛋白mRNA表达和I型胶原蛋白合成。此外，p38 MAPK途径的阻断抑制了TGF-β2诱导的COL1A1和COL1A2启动子活性的增加。
TGF-β2激活p38 MAPK，且p38 MAPK在将TGF-β2信号传递至视网膜色素上皮细胞中I型胶原蛋白生成过程中发挥作用。
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