CHARACTERIZATION OF THE ENDOTHELIUM-SPECIFIC MURINE VASCULAR ENDOTHELIAL GROWTH-FACTOR RECEPTOR-2 (FLK-1) PROMOTER

Flk-1, a high-affinity signaling receptor for vascular endothelial gro wth factor (VEGF), is strongly and specifically expressed on endotheli al cells during embryonic development of the vascular system and durin g tumor angiogenesis. Disruption of Flk-1 gene function has recently b een shown to prevent completely endothelial cell differentiation durin g murine embryonic development. To gain insights into the mechanisms t hat regulate the endothelium-specific Flk-1 expression, we have isolat ed the 5'-flanking region of the murine Flk-1 gene. RNase protection a nd primer extension analyses revealed a single transcriptional start s ite located 299 bp upstream from the translational start site in an in itiator-like pyrimidine-rich sequence. The 5'-flanking region is rich in GC residues and lacks a typical TATA or CAAT box. A luciferase repo rter Construct containing a fragment from nucleotides -1900 to +299 sh owed strong endothelium-specific activity in transfected bovine aortic endothelial cells. Deletion analyses revealed that endothelium-specif ic 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 regulato ry elements were identified between nucleotides -4100 and -623. Two st rong general activating elements were present in the region between nu cleotides -96 and -37, which contains one potential NF kappa B and thr ee potential AP-2 binding sites. This study shows that Flk-1 expressio n in endothelial cells is mainly regulated by an endothelium-specific activating element in the long 5'-untranslated region of the first exo n and by negative regulatory elements located further upstream.