Interplay of Sp1 and Egr-1 in the Proximal Platelet-derived Growth Factor A-Chain Promoter in Cultured Vascular Endothelial Cells

Levon M Khachigian,Amy J Williams,Tucker Collins

doi:10.1074/jbc.270.46.27679

Levon M Khachigian, Amy J Williams + Show 1 more

Open Access

https://doi.org/10.1074/jbc.270.46.27679

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Abstract

The platelet-derived growth factor (PDGF) A-chain has been implicated in the initiation and progression of vascular occlusive lesions. The elements in the human PDGF-A promoter that mediate increased expression of the gene in vascular endothelial cells have not been identified. A potent inducer of PDGF-A expression in endothelial cells is phorbol 12-myristate 13-acetate (PMA). 5'-Deletion and transfection analysis revealed that a G+C-rich region in the proximal PDGF-A promoter is required for PMA-inducible gene expression. This region bears overlapping consensus recognition sequences for Sp1 and Egr-1. PMA induces Egr-1 mRNA expression within 1 h, whereas PDGF-A transcript levels increase after 2-4 h. Constitutive levels of Sp1 are not altered over 24 h. A specific nucleoprotein complex is formed when an oligonucleotide bearing the G+C-rich element is incubated with nuclear extracts from PMA-treated cells. The temporal appearance of this complex is consistent with the transient increase in Egr-1 transcripts. Antibodies to Egr-1 completely supershift the PMA-induced complex. Interestingly, increased nuclear levels of Egr-1 attenuate the ability of Sp1 to interact with the oligonucleotide, implicating competition between Egr-1 and Sp1 for the G+C-rich element. Binding studies with recombinant proteins demonstrate that Egr-1 can displace Sp1 from this region. Insertion of the G+C-rich element into a hybrid promoter-reporter construct confers PMA inducibility on the construct. Mutations that abolish Egr-1 binding also abrogate expression induced by PMA or overexpressed Egr-1. These findings demonstrate that PMA-induced Egr-1 displaces Sp1 from the G+C-rich element and activates expression driven by the PDGF-A proximal promoter in endothelial cells. The Sp1/Egr-1 displacement mechanism may be an important regulatory circuit in the control of inducible gene expression in vascular endothelial cells.

Highlights

§ Established Investigator of the American Heart Association
phorbol 12-myristate 13-acetate (PMA) was used as a model agonist to define specific nuclear transcription factors that interact with functional nucleotide elements in the proximal platelet-derived growth factor (PDGF)-A promoter in cultured vascular endothelial cells
Identification of Regions in the PDGF-A Promoter Required for Optimal Basal Activity and PMA-induced Gene Expression in Endothelial Cells—To define the minimal promoter region that mediates basal expression in cultured endothelial cells, BAEC were transiently transfected with reporter constructs bearing nested 5Ј-deletions of the PDGF-A promoter fused to chloramphenicol acetyltransferase (CAT) cDNA

Summary

Introduction

§ Established Investigator of the American Heart Association. To whom correspondence should be addressed: Vascular Research Division, Dept. of Pathology, Brigham and Women’s Hospital, 221 Longwood Ave., Boston, MA 02115. PMA was used as a model agonist to define specific nuclear transcription factors that interact with functional nucleotide elements in the proximal PDGF-A promoter in cultured vascular endothelial cells. Gel shift and supershift studies demonstrate that both PMA-induced and recombinant Egr-1 bind to the GϩC-rich element by displacing Sp1.

Results

Conclusion