A cAMP Response Element and an Ets Motif Are Involved in the Transcriptional Regulation of flt-1 Tyrosine Kinase (Vascular Endothelial Growth Factor Receptor 1) Gene

Abstract

The flt-1 gene encodes a transmembrane tyrosine kinase, Flt-1, a receptor for vascular endothelial growth factor. The expression of flt-1 gene is restricted to endothelial cells in vivo. To understand the molecular mechanism underlying endothelial-specific expression of this gene, we studied the functional significance of transcriptional motifs in the 200-base pair region of the human flt-1 gene promoter, which has been identified to confer cell type specificity. By point mutation analysis using chloramphenicol acetyltransferase plasmids in 293E1 cells, which express significant levels of flt-1 mRNA, we found that an Ets motif, E4, at ؊54 to ؊51 and a cAMP response element (CRE) at ؊83 to ؊76 are involved in the transcriptional regulation of this gene. Disruption of either this CRE or E4 within the promoter sequence of 90 base pairs resulted in a decrease in chloramphenicol acetyltransferase activity of 90%, indicating that co-existence of both of CRE and Ets motif E4 is necessary for transcription of the flt-1 gene. Co-transfection of an expression vector containing c-ets-1, c-ets-2, or c-erg cDNA with this 90-base pair sequence yielded a 5-8-fold elevation of chloramphenicol acetyltransferase activity, further supporting the idea that Ets family protein(s) participates in the regulation of the flt-1 gene. Gel shift assays using nuclear extracts of 293E1 and endothelial cells demonstrated the existence of protein factor(s) that specifically binds to CRE and Ets motif E4, respectively. Taken together, our results strongly suggest cooperation of a CRE and an Ets motif for the function of the flt-1 gene promoter.

Basal Transcription of the Mouse Sarco(endo)plasmic Reticulum Ca2+-ATPase Type 3 Gene in Endothelial Cells Is Controlled by Ets-1 and Sp1

“…This situation is similar to that observed with several endothelial genes where EBS have been shown to be critical for activity of the promoter, such as the mouse VE-cadherin gene (40), Tie-1 (41), Tie-2 (34), Flt-1 (42), and Flk-1 (33), and raised the question of the identification of the ETS factor(s) involved in this regulation. The ETS family contains ϳ30 transcriptional activators and repressors that share a common DNA binding domain (30).…”

Ets transcription factors bind and transactivate the core promoter of the von Willebrand factor gene

“…Many promoters were described for which a single site, among several in tandem Ets binding motifs, is su cient for binding Ets proteins and for transcriptional activation (Wakiya et al, 1996;Rosen et al, 1994). In these cases, as well as when a single Ets binding site is present, binding of cooperative transcription factor(s) is necessary for speci®c transactivation.…”

“…Cultured HUVEC contain low transcript levels for c-Ets-1 and c-Ets-2 and high level for Erg (Khachigian et al, 1994). Recent studies have shown the essential role of Ets binding sites in basal promoters of several genes expressed in endothelial cells (Khachigan et al, 1994;von der Ahe et al, 1993;Wakiya et al, 1996).…”

ETS1 lowers capillary endothelial cell density at confluence and induces the expression of VE-cadherin

“…This binding domain recognizes the core sequence GGAA/T, named the Ets-binding site (EBS) (Gunther et al, 1990). In vitro, Ets factors transactivate the promoters of several genes that are important in angiogenesis such as the genes encoding VEGF-R1 (Wakiya et al, 1996), the platelet-derived growth factor-B chain (Khachigian et al, 1994), the a4 integrin sub-unit (Rosen et al, 1994) and the receptor Tie (Iljin et al, 1999). A few Ets1 target genes have been already identi®ed by analysis of endogenous transcripts in Ets1-over-expressing cells.…”