Choi Jongkyu, Park Sun Young, Joo Choun-Ki
Department of Ophthalmology and Visual Science, College of Medicine, The Catholic University of Korea, Seoul, Korea.
Invest Ophthalmol Vis Sci. 2007 Jun;48(6):2708-18. doi: 10.1167/iovs.06-0639.
TGFbeta is a potent candidate for epithelial-mesenchymal transition (EMT) during the development of anterior polar cataracts in the human lens. The Snail superfamily is involved in EMT through the repression of E-cadherin production. This study was conducted to determine whether the Snail gene family is activated in the process of TGFbeta1-induced EMT and how TGFbeta1 regulates the expression of this gene family.
Total RNA extracted from human cataract samples was subjected to the real-time PCR quantification of Slug mRNA. Induction of Slug expression by TGFbeta1 (10 ng/mL) in lens epithelial cells was determined by RT-PCR, immunostaining, immunoblot analysis, and Slug promoter analysis. A series of Slug promoter deletion constructs was used to identify the putative regulatory element responsive to TGF signaling. Chromatin immunoprecipitation was performed to determine whether Sp1 associates with the endogenous Slug promoter. Inhibition of Slug expression with Slug siRNA was used to investigate the role of Slug in TGFbeta-mediated EMT.
Slug levels were highly upregulated in lens epithelial cells obtained from patients with anterior polar cataracts. Treatment of TGFbeta1 induced the expression of Slug in both lens and other epithelial cells in vitro. TGFbeta1-induced Slug expression was significantly inhibited by the MEK- and JNK/SAPK-specific inhibitors, but not by transfection with dominant-negative forms of Smads or small GTPase proteins, indicating that MAPK pathways are involved in the regulation of Slug expression by TGFbeta1. The Slug promoter analysis revealed that the Sp1 binding site in the Slug promoter is responsible for TGFbeta1-induced Slug expression. In addition, the TGFbeta1-mediated repression of E-cadherin was significantly inhibited by Slug siRNA.
These data suggest that TGFbeta1 induces Slug expression and that the repression of E-cadherin production by TGFbeta1 is mediated by the induction of Slug in lens epithelial cells.
转化生长因子β（TGFβ）是人类晶状体前极性白内障发育过程中上皮-间质转化（EMT）的一个有力候选因子。Snail超家族通过抑制E-钙黏蛋白的产生参与EMT过程。本研究旨在确定Snail基因家族在TGFβ1诱导的EMT过程中是否被激活，以及TGFβ1如何调节该基因家族的表达。
从人类白内障样本中提取的总RNA用于Slug mRNA的实时PCR定量分析。通过RT-PCR、免疫染色、免疫印迹分析和Slug启动子分析，确定TGFβ1（10 ng/mL）对晶状体上皮细胞中Slug表达的诱导作用。使用一系列Slug启动子缺失构建体来鉴定对TGF信号有反应的假定调控元件。进行染色质免疫沉淀以确定Sp1是否与内源性Slug启动子结合。用Slug siRNA抑制Slug表达以研究Slug在TGFβ介导的EMT中的作用。
在前极性白内障患者的晶状体上皮细胞中，Slug水平高度上调。TGFβ1处理在体外诱导晶状体和其他上皮细胞中Slug的表达。MEK和JNK/SAPK特异性抑制剂显著抑制TGFβ1诱导的Slug表达，但用显性负性形式的Smads或小GTPase蛋白转染则无此作用，这表明丝裂原活化蛋白激酶（MAPK）途径参与TGFβ1对Slug表达的调节。Slug启动子分析显示，Slug启动子中的Sp1结合位点负责TGFβ1诱导的Slug表达。此外，Slug siRNA显著抑制TGFβ1介导的E-钙黏蛋白的抑制作用。
这些数据表明，TGFβ1诱导Slug表达，并且TGFβ1对E-钙黏蛋白产生的抑制作用是由晶状体上皮细胞中Slug的诱导介导的。
