Myc is an essential negative regulator of platelet-derived growth factor beta receptor expression.

Platelet-derived growth factor BB (PDGF BB) is a potent mitogen for fibroblasts as well as many other cell types. Interaction of PDGF BB with the PDGF beta receptor (PDGF-betaR) activates numerous signaling pathways and leads to a decrease in receptor expression on the cell surface. PDGF-betaR downregulation is effected at two levels, the immediate internalization of ligand-receptor complexes and the reduction in pdgf-betar mRNA expression. Our studies show that pdgf-betar mRNA suppression is regulated by the c-myc proto-oncogene. Both constitutive and inducible ectopic Myc protein can suppress pdgf-betar mRNA and protein. Suppression of pdgf-betar mRNA in response to Myc is specific, since expression of the related receptor pdgf-alphar is not affected. We further show that Myc suppresses pdgf-betar mRNA expression by a mechanism which is distinguishable from Myc autosuppression. Analysis of c-Myc-null fibroblasts demonstrates that Myc is required for the repression of pdgf-betar mRNA expression in quiescent fibroblasts following mitogen stimulation. In addition, it is evident that the Myc-mediated repression of pdgf-betar mRNA levels plays an important role in the regulation of basal pdgf-betar expression in proliferating cells. Thus, our studies suggest an essential role for Myc in a negative-feedback loop regulating the expression of the PDGF-betaR.

Myc represses the growth arrest gene gadd45.

The c-Myc protein strongly stimulates cellular proliferation, inducing cells to exit G0/G1 and enter the cell cycle. At a molecular level, Myc prevents growth arrest and drives cell cycle progression through the transcriptional regulation of Myc-target genes. Expression of the growth arrest and DNA damage inducible gene 45 (gadd45) is elevated in response to DNA damaging agents, such as ionizing radiation via a p53-dependent mechanism, upon nutrient deprivation, or during differentiation. Gadd45 holds a vital role in growth arrest as ectopic expression confers a strong block to proliferation. Exposure of quiescent cells to mitogen stimulates a rapid increase in c-Myc expression which is followed by the subsequent reduction in gadd45 expression. The kinetics of these two regulatory events suggest that Myc suppresses the expression of gadd45, contributing to G0/G1 phase exit of the cell cycle. Indeed, ectopic Myc expression in primary and immortalized fibroblasts results in the suppression of gadd45 mRNA levels, by a mechanism which is independent of cell cycle progression. Using an inducible MycER system, rapid suppression of gadd45 mRNA is first evident approximately 0.5 h following Myc activation. The reduction in gadd45 mRNA expression occurs at the transcriptional level and is mediated by a p53-independent pathway. Moreover, Myc suppression and p53 induction of gadd45 following exposure to ionizing radiation are non-competitive co-regulatory events. Myc suppression of gadd45 defines a novel pathway through which Myc promotes cell cycle entry and prevents growth arrest of transformed cells.

The Myc negative autoregulation mechanism requires Myc-Max association and involves the c-myc P2 minimal promoter.

Increasing evidence supports an important biological role for Myc in the downregulation of specific gene transcription. Recent studies suggest that c-Myc may suppress promoter activity through proteins of the basal transcription machinery. We have previously reported that Myc protein, in combination with additional cellular factors, suppresses transcription initiation from the c-myc promoter. To characterize the cis components of this Myc negative autoregulation pathway, fragments of the human c-myc promoter were inserted upstream of luciferase reporter genes and assayed for responsiveness to inducible MycER activation in Rat-1 fibroblasts. We found four- to fivefold suppression of a c-myc P2 minimal promoter fragment upon induction of wild-type MycER protein activity, while induction of a mutant MycER protein lacking amino acids 106 to 143 required for Myc autosuppression failed to elicit this response. This assay is physiologically significant, as it reflects Myc autosuppression of the endogenous c-myc gene with regard to kinetics, dose dependency, cell type specificity, and c-Myc functional domains. Analysis of mutations within the P2 minimal promoter indicated that the cis components of Myc autosuppression could not be ascribed to any known protein-binding motifs. In addition, to address the trans factors required for Myc negative autoregulation, we expressed MycEG and MaxEG leucine zipper dimerization mutants in Rat-1 cells and found that Myc-Max heterodimerization is obligatory for Myc autosuppression. Two models for the Myc autosuppression mechanism are discussed.

Loss of Rb and Myc activation co-operate to suppress cyclin D1 and contribute to transformation.

Cyclin D1 can bind and phosphorylate the product (pRb) of the retinoblastoma gene (RB-1) and recent evidence suggests pRb, in turn, may regulate cyclin D1 protein expression. In transformed cell lines, loss of pRb activity strongly correlates with a decrease in cyclin D1 protein expression, and conversely, introduction of pRb can induce cyclin D1 promoter activity. We show here that pRb does not regulate cyclin D1 directly as basal and serum-stimulated levels of cyclin D1 protein and kinase activity are similar in wildtype and pRb-deficient primary mouse embryonic fibroblasts (MEFs). These observations suggest that the suppression of cyclin D1 in pRb-minus tumour cell lines requires both loss of pRb and at least one additional genetic event. We have determined that constitutive, ectopic Myc expression in pRb-deficient, but not wildtype, MEFs suppresses cyclin D1 protein expression and kinase activity. Regulation is evident at either the level of RNA or protein expression. Phenotypically, pRb-deficient MEFs consistently exhibited a delayed growth response in comparison to wildtype MEFs. This growth delay is abrogated in pRb-deficient MEFs which are expressing ectopic Myc protein, coincident with the loss of cyclin D1 protein expression. Moreover, these cells exhibit an increased proliferative capacity, and they no longer show contact inhibition. Our results support a cross-regulatory mechanism between Myc, pRb and cyclin D1 and suggest a novel role for cyclin D1 in tumorigenesis.

Myc induces cyclin D1 expression in the absence of de novo protein synthesis and links mitogen-stimulated signal transduction to the cell cycle.

Mitogen-activated signal transduction frequently leads to the induction of the c-myc proto-oncogene, but the subsequent molecular events downstream of Myc protein expression which promote cell cycle progression remain unclear. To study Myc-specific effects, without the complexity of the broader proliferative response evoked by serum, we employed the MycER-inducible system in the non-transformed Rat-1 cell line. We demonstrate that activation of wild-type, but not mutant, MycER is sufficient to transiently induce cyclin D1 RNA as well as protein expression to physiological levels, and promote G0/G1 to S phase transition of the cell cycle. Stimulation of endogenous cyclin D1 RNA is rapid and clearly evident within 30 min of MycER-activation, reaching a peak at 3 h. Nuclear run-on analysis demonstrates that this induction occurs at the transcriptional level with a fivefold increase in the rate of transcription. Moreover, MycER induces cyclin D1 transcription with equal efficacy in the presence or absence of de novo protein synthesis. Our work shows that Myc and cyclin D1 lie consecutively in a major proliferation-control pathway, and together create a pivotal connection between signal transduction and cell cycle control.