Novel negative regulatory element in the platelet-derived growth factor B chain promoter that mediates ERK-dependent transcriptional repression.
Platelet-derived growth factor (PDGF), which consists of an A and/or B chain, stimulates migration and proliferation in vascular smooth muscle cells as well as a large number of other cell types. Investigations over recent years have defined roles for several positive regulatory transcription factors in the PDGF-B promoter. However, little is known about the transcriptional mechanisms that negatively regulate this gene. Here, we used transient transfection and 5' deletion analysis to define a specific region in the PDGF-B promoter-mediating repression in vascular smooth muscle cells. Gel retardation assays revealed this region is bound by nuclear protein(s) in a specific manner. Supershift assays excluded the direct association of Sp1, Sp3, and Egr-1. Mutation of the negative regulatory element no longer supported nucleoprotein complex formation and, when introduced into the PDGF-B promoter, rescued the promoter from repression. Promoter activity was also restored by transfection of oligonucleotide decoys bearing the repressor binding site. The MEK1/2 inhibitor, PD98059, and a dominant negative construct generating inactive ERK1 increased reporter expression driven by the PDGF-B promoter. In contrast, the MEK inhibitor had no effect on the activity of the mutant PDGF-B promoter. These effects were cell type-specific, since neither suppression of the PDGF-B promoter nor nucleoprotein complex formation was observed in vascular endothelial cells. These findings define a distinct negative regulatory element in the PDGF-B promoter that interacts with nuclear protein(s) and inhibits PDGF-B promoter-dependent gene expression in an ERK-dependent manner.
Rafty
L A
LA
Centre for Thrombosis and Vascular Research, The University of New South Wales, Department of Haematology, Prince of Wales Hospital, Sydney, Australia.
Khachigian
L M
LM
D001483:Base Sequence; D002478:Cells, Cultured; D020928:Mitogen-Activated Protein Kinases / Q000502:physiology*; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000166:cytology; D010982:Platelet-Derived Growth Factor / Q000235:genetics*; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000502:physiology*
D010982:Platelet-Derived Growth Factor; D012097:Repressor Proteins; D020928:Mitogen-Activated Protein Kinases