Requirement of a GT Box (Sp1 Site) and Two Ets Binding Sites for Vascular Endothelial Cadherin Gene Transcription

Abstract

Vascular endothelial cadherin (VE cadherin) gene encodes a Ca2؉ -dependent cell adhesion molecule required for the organization of interendothelial junctions. This gene is exclusively and constitutively expressed in endothelial cells. Previous data with transgenic mice revealed that the transcriptional regulatory elements present within a ؊2486/؉24 DNA fragment of mouse VE cadherin gene mimic the tissue-specific activity of the endogenous promoter. In this study, we analyzed elements implicated in the function of the proximal regulatory region. Electrophoretic mobility shift assay identified a GT-rich sequence (positions ؊49/؊39) interacting with factors related to the Sp1 family. Point mutations abolished the binding of nuclear proteins in vitro and drastically diminished the activity of the promoter in transient transfection assay. Supershift assays with antibodies against proteins of the Sp1 family revealed that Sp1 and Sp3 interact with this region of the VE cadherin promoter. Furthermore, two GGAA 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 binding sites are essential for promoter activity. In vitro binding assays in the presence of various antisera suggest that Erg is one of the proteins interacting with the ؊109/؊106 site.

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

“…Since the double EBS2/EBS4 mutant did not respond to Ets1 anymore, there must be no additional Ets1-responding sites left in the rest of the promoter. A previous study using a shorter version of the promoter had shown that EBS2 and EBS4 were necessary to the basal transcriptional activity of the promoter in endothelial cells, and that EBS4 was more e ective than EBS2 for this activity (Gory et al, 1998). Here, we suggest that Ets1 could be the Ets member that mediates these e ects: (i) EBS2 and EBS4 are necessary for transactivation by Ets1, (ii) transactivating activities of EBS2 and EBS4 are additive in response to Ets1, (ii) EBS4 is more responsive to Ets1 than EBS2, this latter e ect being probably due to its better binding to rEts1 than EBS2 (Figure 7a).…”

“…This approach was chosen primarily because endothelial cells are notoriously refractory to transfection and our experiments validated this choice: the MFG retrovirus transduced the gene to the cells with more than 80% e ciency and the viral promoter provided signi®cantly higher expression levels of Ets1 transcripts and proteins in Figure 7 Binding of Ets1 to functional EBS in the VE-cadherin gene. (A) Recombinant Ets1 (rEts1, approximately 1 ng) was assayed for binding to 32 P-labeled double-stranded oligonucleotides (10 ng) corresponding to the various EBS present in the proximal region of the VE-cadherin gene (Gory et al, 1998) by electromobility-shift assay. The complexes formed by rEts1 and EBS2 or EBS4 were super-shifted by the addition of 250 ng of anti-Ets1 antibody (SS).…”

“…Several EBS are present in the promoter of the VEcadherin gene (Huber et al, 1996;Gory et al, 1998). In order to analyse the putative role of Ets1 in the control of this promoter, we performed transactivation experiments using an Ets1-expression plasmid and the 72486CAT VE-cadherin gene reporter vector, which contains the 72486/+24 promoter sequence of VEcadherin placed upstream a chloramphenicol acetyl transferase gene .…”

“…Transient transfection of the pMFGTagEts1 vector in 3T3 cells induced a 4.4+0.2-fold activation of the 72486CAT reporter vector over pBLCAT3 control. In order to identify the EBS present in this region that are involved in the regulation of the protein gene by Ets1, we produced a recombinant Ets1 (rEts1) and analysed its binding to ®ve EBS among which two (EBS2 and EBS4) are known to be necessary for gene expression (Gory et al, 1998). rEts1 bound to EBS2 and to EBS4 and to the control MSV-LTR EBS ( Figure 7A) but not to EBS1, EBS3 or EBS5.…”

“…The 72486/+24 region of the VE-cadherin gene promoter drives the speci®c expression of the gene in endothelial cells . Several EBS have been identi®ed in the 7139/+24 region; two are essential for transcriptional activity of the promoter in ®broblasts and endothelial cells (Gory et al, 1998). The Etsfamily members that bind to the proximal promoter and the transcription factors that mediate endothelial speci®city have not been identi®ed so far.…”