Transcriptional repression of the RET proto-oncogene by a mitogen activated protein kinase-dependent signalling pathway
Introduction
The RET proto-oncogene encodes a receptor tyrosine kinase required for the normal development of tissues derived from the neural crest and urogenital systems. Ligands which belong to the glial-cell line derived neurotrophic factor family form complexes with their glycosyl-phosphatidylinositol-anchored co-receptors and transduce their signal through RET (Saarma and Sariola, 1999). Constitutional RET mutations are associated with the multiple endocrine neoplasia type 2 syndrome (MEN 2) which is characterised by medullary thyroid carcinoma (MTC), a tumour originating in thyroid C-cells and the most frequent malignancy of MEN 2 (for review see Hansford and Mulligan, 2000).
During embryogenesis in both mouse and rat, ret is detected in neural crest cells which give rise to the thyroid C-cells, adrenal medulla, and intestinal autonomic ganglia (Pachnis et al., 1993). The temporal and spatial pattern of expression of this gene during development suggests that it is tightly regulated at the level of transcription. In adult tissues, the level of RET expression appears to be low to absent which may indicate that RET is transcriptionally silenced (Takaya et al., 1996). High levels of RET transcripts are detected in MTC (Santoro et al., 1990). The mechanism for the relatively high levels of RET expression, in the absence of obvious RET mutation in these specific tumour types, remains unclear. However, the relative levels of critical transcription factors required for RET expression and their state of activation in these different tissues may contribute to the transformation process.
The human RET proto-oncogene spans approximately 55 kb and has been shown to have a single transcription start site (Itoh et al., 1992, Andrew et al., 2000). The RET promoter is TATA-less and highly GC-rich (Itoh et al., 1992); (Andrew et al., 2000). A previous study from our laboratory has characterised RET basal promoter activity in the MTC cell line TT, and shown that it is dependent on sephacryl and phosphocellulose protein 1 (Sp1) and Sp3 binding within this region (Andrew et al., 2000). The highly GC-rich sequence of the RET proximal promoter suggested that methylation might account for the repression seen in most adult tissues. Surprisingly, analysis of this region failed to identify any methylation (Munnes et al., 1998). As Sp1 binding has been shown to prevent methylation in a number of promoters (Holler et al., 1988), a certain low level of expression of RET in most tissues may be achieved by the blocking of methylation by Sp1.
The Sp transcription factors are now thought to be part of a larger family of transcription factors containing a highly conserved DNA-binding domain (for review see Turner and Crossley, 1999). Specificity of individual members is achieved by their relative abundance within various cell types. For example, in primary keratinocytes, Sp3 levels exceed those of Sp1 but that ratio is reversed upon differentiation (Apt et al., 1996). Post-translational modification is also known to alter the affinity of Sp1 for its target sequence (Jackson et al., 1990). Interaction with other transcription factors, particularly those with overlapping cis sites, is another obvious source of specificity. Further study of the RET basal promoter sequence identified a potential recognition sequence for early growth response gene 1 (Egr-1), also known as Krox-24; Zif-268; Tis8 and NGF1-A. The Egr-1 site overlaps one of the Sp1 sites previously identified as necessary for basal transcription of RET (Andrew et al., 2000).
The Egr-1 transcription factor is a member of the immediate-early gene group, which includes c-fos and c-jun, and which exhibits de novo transcription within 30 min after mitogenic stimulation of a variety of cell types (Sukhatme et al., 1988). The Egr-1 gene encodes an 82 kDa protein which, like Sp1, contains three zinc finger motifs of the Cys2-His2 type. The consensus binding site for Egr-1 has been defined as 5′-GCGGGGGCG-3′ (Cao et al., 1993). Egr-1 can act either as a positive (Zhang et al., 2001) or negative (Bahouth et al., 2002) regulator of gene transcription, depending upon the presence and proximity of other DNA binding sites, because Egr-1 contains both transactivation and repression domains (Gashler et al., 1993). Overexpression of Egr-1 has been found to suppress transformed growth in a number of cell types (Huang et al., 1995). However, Egr-1 also has an important role in the normal differentiation of many cell types, and can be induced by factors which play a role in maintaining differentiation.
In MTC, silencing of RET expression has been associated with endocrine differentiation and a decrease in cell growth (Carson et al., 1995). Furthermore, this decrease in RET expression is associated with stimulation of Raf-1 in TT, a cell line derived from a patient with aggressive MTC (Carson et al., 1995). Phorbol 12-myristate 13-acetate (PMA), a known inducer of Egr-1, has been previously reported to promote differentiation in the TT cell line (deBustros et al., 1986). In this study, we have used luciferase reporter gene transfections and electrophoretic mobility shift analysis (EMSA) to further elucidate the molecular mechanisms responsible for RET expression in MTC. We demonstrate that a complex of proteins, including Sp1, Sp3, and Egr-1, bind to overlapping sites in the RET promoter to regulate RET expression.
Section snippets
Oligonucleotide synthesis, purification, and labelling
Oligonucleotides were synthesised by Pacific Oligos (Lismore, Australia). Double-stranded oligonucleotides were labelled with [γ-32P]-dATP (Amersham Pharmacia Biotech, Piscataway, NJ, USA) using T4 polynucleotide kinase (New England Biolabs, Beverly, MA) and purified using G-50 Sephadex columns (Amersham Pharmacia Biotech, Piscataway, NJ, USA).
Western blot analysis
Whole cell lysates were prepared by lysing with ice-cold phosphate buffered saline (PBS; 5.8 mM Na2HPO4, 1.7 mM NaH2PO4, 6.8 mM NaCl), containing 1%
PMA-induced alteration in cellular morphology
The MTC cell line TT, grows in monolayers and exhibits heterogeneity in cell size and shape (Leong et al., 1981). Treatment of TT cells with 10−8 M PMA caused a marked alteration in cellular morphology within 3 h. The cells adopted a pavement-like appearance, more normally associated with epithelial cells, and also exhibited partial retraction of neurites. This effect was maintained for at least 48 h in the presence of PMA. No morphological effect was seen with the vehicle control over the same
Discussion
The present study extends our previous findings that RET basal expression is controlled by the transactivators Sp1 and Sp3 in TT cells (Andrew et al., 2000). In this study, we suggest that induction of Egr-1 in TT cells by PMA represses RET expression by displacing the transactivators Sp1 and Sp3 from the minimal promoter. Several findings support this model. First, Egr-1 mRNA is induced in TT cells within minutes of exposure to PMA (Fig. 2D) preceding the appearance of complex C3 on the RET
Acknowledgements
We thank Dr V. Sukhatme (Beth Israel Hospital, Boston) for the Egr-1 cDNA probe, Dr E. Adamson for the recombinant Egr-1 and the Egr-1 expression vector (pCMV-Egr-1) and Dr C. Clarke for the 36B4 cDNA probe. DJM is a R.D. Wright Fellow (NH&MRC, Australia).
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Present address: Developmental Neurobiology Unit, Childrens’ Medical Research Institute, Westmead, NSW 2145, Australia.