Abstract
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.
Highlights
Evidence for the Existence of a Negative Regulatory Element in the Proximal Platelet-derived growth factor (PDGF)-B Promoter—WKY12–22 pup SMCs, which express PDGF-B mRNA [21], were transiently transfected with a series of chloramphenicol acetyltransferase (CAT) reporter constructs driven by various-sized fragments of the PDGF-B promoter (Fig. 1, upper panel)
Basal CAT activity was readily detectable and not significantly different in SMCs transfected with constructs d77 and d26, bearing 82 and 153 bp of PDGF-B promoter sequence, respectively (Fig. 1, lower panel)
Nuclear Protein(s) Interact with the Negative Regulatory Element in the Proximal PDGF-B Promoter—We performed Electrophoretic mobility shift assay (EMSA) to gain insight into the mechanisms underlying the Sequences are shown in 5Ј 3 3Ј direction
Summary
Mutational, decoy, and transient transfection analysis, we have identified a novel negative regulatory element in the PDGF-B promoter that mediates repression of the promoter in a sequence-specific and MEK/ERK-dependent manner. Evidence for the Existence of a Negative Regulatory Element in the Proximal PDGF-B Promoter—WKY12–22 pup SMCs, which express PDGF-B mRNA [21], were transiently transfected with a series of CAT reporter constructs driven by various-sized fragments of the PDGF-B promoter (Fig. 1, upper panel).
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