Long-term, heterologous down-regulation of the epidermal growth factor receptor in PC12 cells by nerve growth factor.

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...

mentioning

confidence: 99%

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

Liu¹,

Gong²,

Guo³

et al. 2001

“…PC12 cells treated with NGF exhibit an attenuated response to EGF (9) because EGF receptors are down-regulated (9,11). The cellular mechanisms that mediate NGF-induced EGF receptor down-regulation are unknown and represent an interesting question in neuronal development.…”

mentioning

confidence: 99%

“…Cell Culture-PC12 cells were grown in Dulbecco's modified Eagle's medium (DMEM; Life Technologies, Inc.) supplemented with 7% fetal bovine serum, 7% horse serum, 100 g/ml streptomycin, and 100 units/ml penicillin, all from Life Technologies, Inc. (11). In all experiments involving extended treatment with NGF, the medium was changed, and fresh NGF was added every other day.…”

mentioning

confidence: 99%

Down-regulation of Epidermal Growth Factor Receptors by Nerve Growth Factor in PC12 Cells Is p140 -, Ras-, and Src-dependent

Lazarovici

Oshima

Shavit

et al. 1997

PC12 cells, derived from a rat pheochromocytoma, have been extensively used as a model of neuronal differentiation (1), because the cells acquire the phenotype of sympathetic neurons and stop dividing in response to nerve growth factor (NGF) 1 (2).In PC12 cells, NGF interacts with two distinct plasma membrane receptor proteins: p75 NGFR , a cysteine-rich glycoprotein having a relatively low affinity for NGF (3), and p140 trk , a receptor tyrosine kinase activated by NGF, which binds NGF with high affinity and mediates many of the biological activities of this neurotrophin (4). The binding of NGF to the p140 trk receptor stimulates rapid tyrosine autophosphorylation of the receptor and activation of several signal-transducing proteins (4). Activated p140 trk receptors bind to and tyrosine phosphorylate the signaling substrates phospholipase C␥1, phosphatidylinositol-3 kinase, and Src homologous collagen protein (4), resulting in their activation. The latter stimulates Ras activity which subsequently activates a series of serine/threonine kinases including B-Raf, myelin basic protein kinase kinase, the Erks, and p90 rsk (4). This Ras-Erk pathway plays a major role in the activation of transcriptional events by NGF and in NGFinduced neuronal differentiation (5), as illustrated by inhibition of NGF actions upon expression of dominant-negative HaRas (Asn-17) proteins (6, 7). In addition a role for pp60 c-src in NGF actions has been suggested (5) by experiments involving the expression of a dominant-interfering kinase-inactive Src (8).PC12 cells also express plasma membrane receptors for epidermal growth factor (EGF) (9, 10), a mild mitogen for these cells (9). PC12 cells treated with NGF exhibit an attenuated response to EGF (9) because EGF receptors are down-regulated (9, 11). The cellular mechanisms that mediate NGF-induced EGF receptor down-regulation are unknown and represent an interesting question in neuronal development. The existence of functional receptors for both NGF and EGF on PC12 cells and the recent advances in understanding the signaling pathways activated by these receptors make these cells a useful model for the study of cross-regulation between NGF and EGF receptors during differentiation.In this study, we have used a number of PC12 variant cell lines with different levels of p140 trk (12, 13) and EGF receptor expression and dominant-negative Ras (7,14,15) or Src (5,6,8) PC12 transfectants in an attempt to elucidate the molecular mechanisms of the receptor cross-talk required by NGF to down-regulate EGF receptors. Our findings implicate the involvement of p140 trk -, Ras-, and Src-dependent signaling pathways in NGF-induced down-regulation of EGF receptors while * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ‡ Permanent address:

“…NGF-induced down-regulation of the EGFR is under transcriptional control (17), and that control is p140trk-, Ras-, and Src-dependent (16). The detailed cellular and molecular mechanisms that mediate NGF-induced EGFR down-regulation are unknown.…”

mentioning

confidence: 99%

“…Our previous studies (16,17) have shown that treatment of PC12 cells with NGF causes a profound down-regulation of both EGFR mRNA and protein. NGF-induced down-regulation of the EGFR is under transcriptional control (17), and that control is p140trk-, Ras-, and Src-dependent (16).…”

mentioning

confidence: 99%

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

Liu¹,

Katagiri²,

Jiang³

et al. 2000

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.