Characterization of the Endothelium-Specific Murine Vascular Endothelial Growth Factor Receptor-2 (Flk-1) Promoter

Abstract

Flk-1, a high-affinity signaling receptor for vascular endothelial growth factor (VEGF), is strongly and specifically expressed on endothelial cells during embryonic development of the vascular system and during tumor angiogenesis. Disruption of Flk-1 gene function has recently been shown to prevent completely endothelial cell differentiation during murine embryonic development. To gain insights into the mechanisms that regulate the endothelium-specific Flk-1 expression, we have isolated the 5′-flanking region of the murine Flk-1 gene. RNase protection and primer extension analyses revealed a single transcriptional start site located 299 bp upstream from the translational start site in an initiator-like pyrimidine-rich sequence. The 5′-flanking region is rich in GC residues and lacks a typical TATA or CAAT box. A luciferase reporter construct containing a fragment from nucleotides −1900 to +299 showed strong endothelium-specific activity in transfected bovine aortic endothelial cells. Deletion analyses revealed that endothelium-specific Flk-1 expression is stimulated by the 5′-untranslated region of the first exon, which contains an activating element between nucleotides +137 and +299. In addition, two endothelium-specific negative regulatory elements were identified between nucleotides −4100 and −623. Two strong general activating elements were present in the region between nucleotides −96 and −37, which contains one potential NFκB and three potential AP-2 binding sites. This study shows that Flk-1 expression in endothelial cells is mainly regulated by an endothelium-specific activating element in the long 5′-untranslated region of the first exon and by negative regulatory elements located further upstream.

A strategy for enhancing the transcriptional activity of weak cell type-specific promoters

“…Other EC promoters are stronger, but less specific, such as the promoters for PECAM-1/CD31 20,21 or flk-1/KDR. 22,23 Another example is the promoter of the sucrase-isomaltase (SI) gene which is highly specific for intestinal cells and gastrointestinal tumors, 24,25 but again is a poor transcriptional activator (see Results below). In the present study, we have chosen the vWF and SI promoters as models for the establishment of a strategy that would be suitable for enhancing the transcriptional activity of weak promoters without affecting their specificity (self-enhancing promoters).…”

Directing oncogenic fusion genes into stem cells via an SCL enhancer

“…Further details of construction of these transgenic vectors are available upon request. The mouse Flk1 promoter (17) and enhancer (18), which, in combination, confer endothelial cell-specific expression, were amplified by PCR and cloned into pGL3-Basic vector. For transient transfection of TEL-TRKC fusion in the luciferase assay, coding sequences of both types of TEL-TRKC fusion gene were subcloned into a pcDNA3 expression vector.…”

The role of AGE–RAGE system in the development of diabetic nephropathy in vivo

“…Construction of transgenic mice and induction of diabetes. RAGETg was produced by introducing a transgene that carryed human RAGE genomic DNA under the control of the murine flk-1 promoter, which acts specifically in EC [10]. RAGETg was crossbred with another transgenic mouse carrying human cDNA for inducible nitric oxide synthase (iNOS) under the control of the insulin promoter (iNOSTg) [11].…”