Liu X W, Gong L J, Guo L Y, Katagiri Y, Jiang H, Wang Z Y, Johnson A C, Guroff G
Section on Growth Factors and Laboratory of Developmental and Molecular Immunity, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.
J Biol Chem. 2001 Feb 16;276(7):5068-73. doi: 10.1074/jbc.M008776200. Epub 2000 Nov 8.
Recently, we characterized the rat epidermal growth factor receptor (EGFR) promoter and demonstrated that TCC repeat sequences are required for the down-regulation of EGFR by nerve growth factor (NGF) in PC12 cells. In this study, we report that the Wilms' tumor gene product WT1, a zinc finger transcription factor, is able to enhance the activity of the rat EGFR promoter in cotransfection assays. Gel mobility shift assays demonstrate that WT1 binds to the TCC repeat sequences of the rat EGFR promoter. Overexpression of WT1 resulted in up-regulation of the expression levels of endogenous EGFR in PC12 cells. Interestingly, NGF down-regulated the expression levels of WT1 and EGFR in PC12 cells, but not in the p140(trk)-deficient variant PC12nnr5 cells or in cells expressing either dominant-negative Ras or dominant-negative Src. Most importantly, we evaluated the inhibitory effect of antisense WT1 RNA on EGFR expression, and we found that antisense WT1 RNA could substantially reduce EGFR repression in either histochemical staining study or immunoblot analysis. These results indicate that NGF-induced down-regulation of the EGFR in PC12 cells is mediated through WT1 and that WT1 may play an important role in the differentiation of nerve cells.
最近，我们对大鼠表皮生长因子受体（EGFR）启动子进行了特性分析，并证明了TCC重复序列是神经生长因子（NGF）在PC12细胞中下调EGFR所必需的。在本研究中，我们报告称，威尔姆斯瘤基因产物WT1，一种锌指转录因子，在共转染实验中能够增强大鼠EGFR启动子的活性。凝胶迁移率变动分析表明WT1与大鼠EGFR启动子的TCC重复序列结合。WT1的过表达导致PC12细胞中内源性EGFR表达水平上调。有趣的是，NGF下调了PC12细胞中WT1和EGFR的表达水平，但在p140（trk）缺陷型变体PC12nnr5细胞或表达显性负性Ras或显性负性Src的细胞中未出现这种情况。最重要的是，我们评估了反义WT1 RNA对EGFR表达的抑制作用，并且发现在组织化学染色研究或免疫印迹分析中，反义WT1 RNA均可显著降低EGFR的抑制作用。这些结果表明，NGF诱导的PC12细胞中EGFR的下调是通过WT1介导的，并且WT1可能在神经细胞分化中起重要作用。
