Dong N, Tang X, Yuan X Y, Song H, Li J
Clinical College of Ophthalmology, Tianjin Medical University, Tianjin Eye Hospital, Tianjin 300020, China.
Zhonghua Yan Ke Za Zhi. 2016 Apr 11;52(4):278-84. doi: 10.3760/cma.j.issn.0412-4081.2016.04.009.
Transforming growth factor-β-activated kinase-1 (TAK1) is thought to play a key role in the initiation of Smad-independent TGF-β signaling. This study investigated the role of TAK1 in the epithelial-mesenchymal transition (EMT) lens epithelial cells.
TAK1 was overexpressed in the HLE B-3 cell line by transfecting TAK1-pcDNA3 and TAK1-binding protein 1 (TAB1)-pcDNA3 plasmids. The expression levels of TAK1, phospho-TAK1, E-cadherin, and fibronectin were detected by Western blot analysis and immunocytofluorescence to analyze the effects of overexpression. The levels of α-SMA and type I collagen were analyzed by real-time PCR. Quantitative data were analyzed by Student's t test or one-way analysis of variance (ANOVA) (multiple comparisons using LSD test).
Western blot analysis showed in the TAK1-pcDNA3 plasmids group, expression of TAK1 proteins (1.00±0.03) with a maximum upregulation of approximately 80% at 24 h than it was in the control group (0.19±0.09)(t=8.02, P< 0.01); Western blot analysis showed in the TAB1-pcDNA3 plasmids group, expression of TAB1 proteins (1.00±0.02) with a maximum upregulation of approximately 78% at 24 h than it was in the control group (0.22±0.08)(t=7.63, P<0.01). The levels of E-cadherin/Beta-actin had significant differences among control, overexpression of TAK1 together with TAB1, overexpression of TAK1, and overexpression of TAB1 (1.00±0.02, 0.12±0.03, 0.98±0.09, 0.92±0.08;F=31.03, P<0.01). The levels of fibronectin/Beta-actin had significant differences among control, overexpression of TAK1 together with TAB1, overexpression of TAK1, and overexpression of TAB1 (0.11±0.03, 1.00±0.05, 0.16±0.04, 0.21±0.05;F=35.12, P<0.01). Overexpression of TAK1 with TAB1 resulted in upregulated expression of fibronectin, and downregulated expression of E-cadherin. The expression of E-cadherin was increased and the expression of fibronectin was decreased by TAK1 siRNA and TAK1 chemical inhibitors in the presence of TGF-β2.
These data reveal that TAK1 can induce the EMT of HLE cells, and the inhibition of TAK1 phosphorylation may be a potential novel therapeutic target for the prevention and treatment of posterior capsule opacification. (Chin J Ophthalmol, 2016, 52: 278-284).
转化生长因子-β激活激酶1（TAK1）被认为在非Smad依赖的TGF-β信号通路起始过程中起关键作用。本研究探讨TAK1在上皮-间质转化（EMT）晶状体上皮细胞中的作用。
通过转染TAK1-pcDNA3和TAK1结合蛋白1（TAB1）-pcDNA3质粒，在HLE B-3细胞系中过表达TAK1。采用蛋白质免疫印迹分析和免疫细胞荧光检测TAK1、磷酸化TAK1、E-钙黏蛋白和纤连蛋白的表达水平，以分析过表达的影响。通过实时PCR分析α-平滑肌肌动蛋白（α-SMA）和I型胶原的水平。定量数据采用Student t检验或单因素方差分析（ANOVA）（使用LSD检验进行多重比较）。
蛋白质免疫印迹分析显示，在TAK1-pcDNA3质粒组中，TAK1蛋白表达量为（1.00±0.03），在24小时时比对照组（0.19±0.09）最高上调约80%（t = 8.02，P < 0.01）；蛋白质免疫印迹分析显示，在TAB1-pcDNA3质粒组中，TAB1蛋白表达量为（1.00±0.02），在24小时时比对照组（0.22±0.08）最高上调约78%（t = 7.63，P < 0.01）。E-钙黏蛋白与β-肌动蛋白的水平在对照组、TAK1与TAB1共过表达组、TAK1过表达组和TAB1过表达组之间存在显著差异（1.00±0.02，0.12±0.03，0.98±0.09，0.92±0.08；F = 31.03，P < 0.01）。纤连蛋白与β-肌动蛋白的水平在对照组、TAK1与TAB1共过表达组、TAK1过表达组和TAB1过表达组之间存在显著差异（0.11±0.03，1.00±0.05，0.16±0.04，0.21±0.05；F = 35.12，P < 0.01）。TAK1与TAB1共过表达导致纤连蛋白表达上调，E-钙黏蛋白表达下调。在存在TGF-β2的情况下,TAK1小干扰RNA和TAK1化学抑制剂使E-钙黏蛋白表达增加，纤连蛋白表达减少。
这些数据表明，TAK1可诱导HLE细胞发生EMT，抑制TAK1磷酸化可能是预防和治疗后囊膜混浊的一个潜在新治疗靶点。（《中华眼科杂志》，2016，52：278 - 284）
