Department of Ophthalmology, Yonsei University, Wonju College of Medicine, Wonju, Korea.
Department of Biomedical Science, Hallym University, Chuncheon, Korea.
Invest Ophthalmol Vis Sci. 2019 Jan 2;60(1):82-92. doi: 10.1167/iovs.18-25376.
Profibrotic activation is essential for pterygium development. In this study, we investigated the role of the mechanistic target of rapamycin (mTOR) in regulating TGF-β1-induced myofibroblastic responses in human pterygium fibroblasts (HPFs) and elucidated the relative contributions of mTOR signaling components.
HPFs were pretreated with the mTOR inhibitors rapamycin and Torin2, and TGF-β1-induced expression of profibrotic markers, including α-smooth muscle actin (α-SMA) and fibronectin, was evaluated. RNA interference-based approaches targeting raptor and rictor, regulatory subunits of mTOR complex 1 (mTORC1) and 2 (mTORC2), respectively, were used to determine the impact of each mTOR complex on HPFs. The contractile phenotype of HPFs was assessed by a collagen gel contraction assay.
The mTOR active-site inhibitor Torin2, which suppresses both mTORC1 and mTORC2 activity in HPFs, inhibited TGF-β1-induced expression of α-SMA and fibronectin. The allosteric inhibitor rapamycin only partially suppressed mTORC1 activity and exhibited a minimal effect on the induction of profibrotic markers. The induction of α-SMA and fibronectin in HPFs was abrogated by RNA interference-mediated knockdown of rictor but was only moderately affected by raptor knockdown. Akt inhibition mimicked the effect of Torin2 and rictor knockdown on myofibroblast differentiation of HPFs. mTOR inhibition potently reduced the contractile ability of HPFs in collagen gel contraction assays.
This study found that mTOR signaling promoted profibrotic activation of HPFs and confirmed the importance of the mTORC2-Akt axis in TGF-β1-induced myofibroblast differentiation. Therefore, our study may open up new avenues for the development of novel therapeutic strategies involving targeting of mTOR signaling to treat pterygium.
纤维增生激活对于翼状胬肉的发展至关重要。在这项研究中，我们研究了机械靶标雷帕霉素（mTOR）在调节人翼状胬肉成纤维细胞（HPFs）中 TGF-β1 诱导的肌成纤维反应中的作用，并阐明了 mTOR 信号成分的相对贡献。
用 mTOR 抑制剂雷帕霉素和 Torin2 预处理 HPFs，评估 TGF-β1 诱导的纤维增生标志物，包括α-平滑肌肌动蛋白（α-SMA）和纤维连接蛋白的表达。采用靶向 mTOR 复合物 1（mTORC1）和 2（mTORC2）的调节亚基 raptor 和 rictor 的 RNA 干扰方法，分别确定每个 mTOR 复合物对 HPFs 的影响。通过胶原凝胶收缩测定评估 HPFs 的收缩表型。
mTOR 活性位点抑制剂 Torin2 抑制 HPFs 中 mTORC1 和 mTORC2 的活性，抑制 TGF-β1 诱导的α-SMA 和纤维连接蛋白的表达。变构抑制剂雷帕霉素仅部分抑制 mTORC1 的活性，对纤维增生标志物的诱导作用很小。HPFs 中 α-SMA 和纤维连接蛋白的诱导作用被 rictor 的 RNA 干扰介导的敲低消除，但 raptor 的敲低仅适度影响。Akt 抑制模拟了 Torin2 和 rictor 敲低对 HPFs 肌成纤维分化的影响。mTOR 抑制在胶原凝胶收缩测定中强烈降低了 HPFs 的收缩能力。
这项研究发现 mTOR 信号促进了 HPFs 的纤维增生激活，并证实了 mTORC2-Akt 轴在 TGF-β1 诱导的肌成纤维分化中的重要性。因此，我们的研究可能为开发涉及靶向 mTOR 信号的治疗翼状胬肉的新型治疗策略开辟新途径。
