Sp1 recognition sites in the proximal promoter of the human vascular endothelial growth factor gene are essential for platelet-derived growth factor-induced gene expression

Abstract

Stimulation of NIH3T3 cells with platelet-derived growth factor (PDGF)-BB enhances expression of vascular endothelial growth factor (VEGF), an endothelial cell-speci®c mitogen and a key mediator of tumor angiogenesis. Here, we identi®ed cis-acting VEGF promoter elements and trans-acting factors which are involved in PDGF-stimulated VEGF expression. By 5'-deletion and transient transfection analysis, a G+C-rich region at 785 to 750 of the human VEGF promoter was shown to be necessary and su cient for both PDGF inducible and basal expression. The region contains three potential recognition sites for Sp1 transcription factors, which overlap with two Egr-1 sites. Mutations that abolish the ability of Sp1 to interact with the VEGF promoter element also abrogate expression induced by PDGF. Mutations of the potential Egr-1 binding sites did not a ect PDGF responsiveness. Gel shift and antibody supershift analyses showed that Sp1 and Sp3 interact constitutively with the VEGF promoter element. Our data strongly suggest that enhanced VEGF gene expression in PDGF-induced NIH3T3 cells is mediated by Sp1 and/or Sp3 transcription factors bound to the 785 to 750 promoter region of the VEGF gene.

Expression of the vascular endothelial growth factor gene is inhibited by p73

“…Moreover, mutation within the Sp 1 binding sites in the promoter fragment, abolished p73-responsiveness, suggesting that the e ect of p73 on the VEGF promoter is mediated by the proximal Sp 1 binding sites in the 785 to 750 promoter region. Interestingly, these potential Sp 1 binding sites have been implicated in the regulation of VEGF expression by the platelet-derived growth factor (PDGF) (Finkenzeller et al, 1997) as well as by the tumor suppressor genes von Hippel Lindau (VHL) (Mukhopadhyay et al, 1997) and p53 (Mukhopadhyay et al, 1995). The results presented here indicate that these Sp 1 binding sites also play a role in p73 mediated repression of the VEGF promoter.…”

“…The p73/VP22 fusion protein shows a molecular weight of approximately 120 kDa on SDS ± PAGE this cell line is well characterized with respect to the e ect of ectopically expressed p73 on transcriptional activation of p53 target genes and cell growth (Jost et al, 1997). Saos-2 cells were transiently cotransfected with a VEGF promoter-luciferase-reporter plasmid pLuc 2068 (Finkenzeller et al, 1997), bearing the VEGF promoter region 72018 to +50, and the p73 expression plasmid pCDNA/p73. As shown in Figure 3, co-transfection of pLuc 2068 with p73 resulted in a strong repression of the VEGF promoter activity.…”

“…To de®ne the regions within the VEGF promoter that confer p73 repression of promoter activity, we used a series of VEGF promoter deletion mutants (Finkenzeller et al, 1997). These 5'-deletion mutants have variable 5'-ends (from 72018 bp in pLuc 2068 to 752 bp in pLuc 102) and an identical 3'-end at bp +50 (relative to the transcriptional start site).…”

“…To further determine whether this promoter region may play a role in down-regulation of promoter activity by p73, the promoter region 785 to 750 was cloned into the heterologous thymidine kinase promoter-luciferase test plasmid pTATA, thus generating pTATA (785/750) (Finkenzeller et al, 1997). As shown in Figure 4b, cells transfected with pTA-TA(785/750) expressed 3.5-fold greater luciferase activity as compared to cells transfected with the control plasmid pTATA.…”

“…The construction of the various VEGF luciferase plasmids, pLuc 2068, pLuc 465, pLuc 135, pLuc 102, pLuc 135/SP 1 mut, pLuc 135/Egr-1 mut, pTATA and pTATA(785/750) was described in detail previously (Finkenzeller et al, 1997). The p73 expression plasmid pcDNA3-p73a containing the p73a cDNA with an amino-terminal hemagglutinin (HA) epitope (Jost et al, 1997) was a gift from Dr D Caput.…”