Recently, we characterized the rat epidermal growth factor receptor (EGFR) promoter and demonstrated that TCC repeat sequences are required for the downregulation 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 dominantnegative 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 NGFinduced 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. The epidermal growth factor receptor (EGFR)1 is a member of the ErbB family of ligand-activated tyrosine kinase receptors, which play a central role in the proliferation, differentiation, and/or oncogenesis of epithelial cells, neural cells, and fibroblasts (1). Some years ago, we reported that PC12 cells respond to both epidermal growth factor (EGF) and nerve growth factor (NGF), but that the response to epidermal growth factor is mitogenic, whereas the response to nerve growth factor leads to differentiation (2). It was of interest to determine what would happen if the cells were exposed to both stimuli at the same time. The answer was that the cells simply did not respond to EGF after they had been treated with NGF, and the reason that they did not respond is that the EGFR is markedly down-regulated by treatment of the cells with NGF (3).Our studies have focused on the molecular mechanism by which NGF down-regulates the EGFR. Previous studies have demonstrated that NGF-induced down-regulation of the EGFR was at the transcriptional level (4) and that the down-regulation is p140 trk -, Ras-, and Src-dependent (5). Recently, we isolated and characterized the rat EGFR promoter region and found that TCC repeat sequences in the promoter region of the rat EGFR are required for the down-regulation of rat EGFR by NGF during the differentiation of PC12 cells (6).WT1, the Wilms' tumor suppressor gene, is located at chromosome locus 11p13 and encodes a zinc finger protein that is one of several transcription factors that have been found to interact with TCC repeat sequences (7-9). Experimental evidence has indicated that WT1 not only plays a role during kidney development but is also involved in the development and homeostasis of other tissues. The products of WT1 have been implicated in va...

Our previous studies have shown that treatment of PC12 cells with nerve growth factor (NGF) causes a profound down-regulation of the epidermal growth factor receptor (EGFR) mRNA and protein. Further, the NGFinduced down-regulation of the EGFR is under transcriptional control. To elucidate the molecular mechanism of this down-regulation we have cloned a 2.7-kilobase sequence from the promoter region of the rat EGFR from a rat P1 library. Six transcriptional start sites were identified by 5-rapid amplification of cDNA ends and primer extension. Sequence analysis showed a 62% overall homology with the human EGFR promoter region. To investigate its transcription, 1.1 kilobases of the 5-flanking sequence were fused to a luciferase reporter gene. This sequence exhibited functional promoter activity in transient transfection experiments with PC12, C6, and CV-1 cells. Treatment of PC12 cells with NGF inhibited promoter activity. By transfection of promoter deletion constructs, a silencer element was found between nucleotides ؊260 and ؊181, and TCC repeat sequences appeared to be at least partially responsible for the down-regulation of the EGFR by NGF. Supportive evidence for the relevance of this sequence was obtained from gel mobility shift assays and by transfection of TCC mutation constructs. Our results demonstrate that TCC repeat sequences are required for the down-regulation of rat EGFR by NGF in PC12 cells and may lead to the identification of the NGF-responsive transcription factors.