Cloning and Characterization of the Promoter Region of the Rat Epidermal Growth Factor Receptor Gene and Its Transcriptional Regulation by Nerve Growth Factor in PC12 Cells

Abstract

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.

The Wilms' Tumor Gene Product WT1 Mediates the Down-regulation of the Rat Epidermal Growth Factor Receptor by Nerve Growth Factor in PC12 Cells

“…3, lanes 3 and 4). In support of this result, mutations in TCC repeat sequences abolished the binding of WT1 in electrophoretic mobility shift analysis as described previously (6). Thus, some of complex A contains WT1 that reacts with anti-WT1.…”

“…Activates the Promoter Activity of the Rat EGFR Promoter-Recently, we isolated and characterized the promoter region of the rat EGFR promoter, and we found that TCC repeat sequences in the promoter region are required for NGFinduced transcriptional down-regulation of EGFR (6). The TCC repeat sequences in the human EGFR promoter have been reported to be a DNA-binding sequence for the Wilms' tumor repressor, WT1 (16).…”

“…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).…”

“…Plasmid Constructs-The rat EGFR promoter-luciferase pRE constructs and the chimeric EGFR reporter pRE-TK-Luc plasmids have been described previously (6). The original WT1 expression vector pCMV-WT1 consisted of the full-length protein coding regions under the control of the CMV immediate-early promoter (14).…”

EphrinA5 acts as a tumor suppressor in glioma by negative regulation of epidermal growth factor receptor

“…EGFR promoter reporter plasmid (pER-Luc) from Dr Alfred C. Johnson (Liu et al, 2000); Expression constructs for hEGFR and hEGFR (Y1045F) mutant were from Dr Yosef Yarden (Levkowitz et al, 1999); pcDNA3.1-HA-Ubiquitin from Dr De Camilli P; CD19-Fc and EphA2-Fc expression vectors from Dr Hans-Christian Aasheim (Finne et al, 2004).…”