REQUIREMENT OF A GT BOX (SP1 SITE) AND 2 ETS BINDING-SITES FOR VASCULAR ENDOTHELIAL CADHERIN GENE-TRANSCRIPTION

Vascular endothelial cadherin (VE cadherin) gene encodes a Ca2+-depend ent cell adhesion molecule required for the organization of interendot helial junctions. This gene is exclusively and constitutively expresse d in endothelial cells. Previous data with transgenic mice revealed th at the transcriptional regulatory elements present within a -2486/+24 DNA fragment of mouse VE cadherin gene mimic the tissue-specific activ ity of the endogenous promoter. In this study, we analyzed elements im plicated in the function of the proximal regulatory region. Electropho retic mobility shift assay identified a GT-rich sequence (positions -4 9/-39) interacting with factors related to the Spl family. Point mutat ions abolished the binding of nuclear proteins in vitro and drasticall y diminished the activity of the promoter in transient transfection as say. Supershift assays with antibodies against proteins of the Spl fam ily revealed that Spl and Sp3 interact with this region of the VE cadh erin promoter. Furthermore, two GC;AA motifs, located at positions -93 /-90 and -109/-106, were shown to interact with nuclear factors. Site- directed mutagenesis of these sequences demonstrated that these Ets bi nding sites are essential for promoter activity. In vitro binding assa ys in the presence of various antisera suggest that Erg is one of the proteins interacting with the -109/-106 site.