Myc Is an Essential Negative Regulator of Platelet-Derived Growth Factor Beta Receptor Expression

Abstract

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 ␤ receptor (PDGF-␤R) activates numerous signaling pathways and leads to a decrease in receptor expression on the cell surface. PDGF-␤R downregulation is effected at two levels, the immediate internalization of ligand-receptor complexes and the reduction in pdgf-␤r mRNA expression. Our studies show that pdgf-␤r mRNA suppression is regulated by the c-myc proto-oncogene. Both constitutive and inducible ectopic Myc protein can suppress pdgf-␤r mRNA and protein. Suppression of pdgf-␤r mRNA in response to Myc is specific, since expression of the related receptor pdgf-␣r is not affected. We further show that Myc suppresses pdgf-␤r 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-␤r mRNA expression in quiescent fibroblasts following mitogen stimulation. In addition, it is evident that the Myc-mediated repression of pdgf-␤r mRNA levels plays an important role in the regulation of basal pdgf-␤r expression in proliferating cells. Thus, our studies suggest an essential role for Myc in a negative-feedback loop regulating the expression of the PDGF-␤R.

p73 Independent of c-Myc Represses Transcription of Platelet-derived Growth Factor β-Receptor through Interaction with NF-Y

“…To obtain nuclear extracts, a total of 4 ϫ 10 5 NIH3T3 cells were seeded in 6-cm dishes. Nuclear protein was extracted from the cells at 0, 4,8,12,24, and 48 h after 10% serum stimulation following 48 h of culture in serum-free medium, nuclear extracts and 4 g of protein were preincubated in 20 l of reaction mixture containing 12 mM Tris-HCl, pH 7.9, 0.6 mM EDTA, 20% glycerol, 1 mM dithiothreitol, 5 mM MgCl 2, 0.1 g/l BSA, 0.1% Nonidet P-40, and 1 mg/ml poly(dI⅐dC). Unlabeled competitors (100ϫ labeled oligo), 32 P-labeled oligonucleotide, and antibodies NF-YA or p73 (0.5 g/reaction) were added in this order.…”

“…After 24 h, half of the cells of each cell type were transfected with 2 g of HAp73 expression plasmid/ dish, and the remaining cells were transfected with 2 g of HApcDNA3 vector alone. The cells were kept in 10% FCS, and RNA was extracted essentially as described (25) at 0, 4,8,12,24, and 36 h after transfection. 3 g of total RNA from each time point was transcribed into cDNA with random primers and Moloney murine leukemia virus reverse transcriptase (Invitrogen) according to the manufacturer's protocol.…”

“…As soon as the ligands diminish or cells become confluent or differentiated, tyrosine phosphatases can be activated, reverting the receptor to its normal status (10,11). As a long term effect, the transcription of the receptor itself can be repressed by increased c-Myc following stimulation by PDGF or serum (1,12). c-myc, one of the target genes of PDGF, leads to proliferative responses of cells and, in turn, down-regulates PDGF ␤-receptor, resulting in the cell cycle-dependent expression of the PDGF ␤-receptor.…”

“…c-myc, one of the target genes of PDGF, leads to proliferative responses of cells and, in turn, down-regulates PDGF ␤-receptor, resulting in the cell cycle-dependent expression of the PDGF ␤-receptor. A Myc-null cell line showed a high and stable level of PDGF ␤-receptor mRNA, which disappeared upon forced expression of c-Myc (12). We have previously shown that c-Myc represses the transcription of the receptor through binding to the NF-Y transcription factor and interferes its activation (1).…”

Functional analysis of the N-terminal domain of the Myc oncoprotein

“…Regulation of these, and other, biological activities has been attributed to Myc's ability to repress and activate the expression of specific target genes (Dang, 1999;Grandori et al, 2000;Oster et al, 2002). Indeed, many Myc-regulated genes are involved in the control of cell growth and proliferation, including the Myc-activated genes cad, odc, cyclin D2 and eIF-4E, and Myc-repressed genes gadd45a, pdgfbr, p15 INK4b and c-myc itself (Penn et al, 1990;Bello-Fernandez et al, 1993;Rosenwald et al, 1993;Miltenberger et al, 1995;Marhin et al, 1997;Bouchard et al, 1999;Perez-Roger et al, 1999;Oster et al, 2000;Staller et al, 2001). As the full complement of Myc target genes is uncovered, the contribution of Myc to these biological processes, as well as the molecular mechanism of target gene regulation, will be revealed.…”