Erg regulates junctional integrity and VE-cadherin expression. (A) HUVECs (105 cells/well) grown on gelatin-coated 6-well plates were transfected with either control GB or Erg-specific GB (100 nM) for 48 hours. Phase-contrast microscopy shows that the typical cobblestone monolayer shown in the control cells (left) has become disrupted in the Erg-inhibited cells (right). Original magnification 20×/0.4 Plan N objective lens (Olympus, Tokyo, Japan). (B) HUVECs (105) grown on gelatin-coated 6-well plates were transfected with either control GB or Erg-specific GB (100 nM) for 48 hours. VE-cadherin mRNA levels were determined from RNA extracts from GB-treated HUVECs using RT-PCR normalized against GAPDH. VE-cadherin mRNA was significantly decreased after Erg inhibition, *P < 0.05. Values are means plus or minus SEM, n = 3. (C) VE-cadherin protein expression was also down-regulated in Erg-GB–treated HUVECs, as determined by SDS-PAGE immunoblotting using antibodies to VE-cadherin, normalized against tubulin. *P < .05. Values are means plus or minus SEM, n = 3. (D) HUVECs grown on gelatin-coated glass coverslips were treated with GB as above. After 48 hours, cells were labeled for Erg using a polyclonal rabbit anti-Erg antibody and for VE-cadherin using a mouse monoclonal anti–VE-cadherin antibody. Erg was visualized with AlexaFluor 488 (green), VE-cadherin with AlexaFluor 555 (red), and nuclei were stained with TOPRO-3 (blue). Scale bar, 100 μm. Objective lens used was Plan-Neofluar 20×/0.5 (Carl Zeiss, Jena, Germany). (E) Genomic DNA sequence of the proximal 5′-region of the human VE-cadherin gene. The transcription initiation site is designated as +1. The positions of the Ets binding sites (EBSs) are boxed. The 2 functional EBSs present in the murine sequence, and conserved in the human,30 are indicated by a double-lined box. Nucleotide sequences corresponding to the oligonucleotides used in the ChIP assay (see panel F) are indicated by arrows. The shaded nucleotides at the 5′ and 3′ ends of the sequence denote the position of oligonucleotides used for PCR amplification of the promoter for subsequent cloning into the pGL4 luciferase reporter plasmid. (F) Chromatin immunoprecipitation from a confluent HUVEC monolayer was performed as described in “Methods.” Genomic DNA obtained after immunoprecipitation, using a rabbit anti-Erg polyclonal antibody or a negative control rabbit IgG, was used as template in a PCR reaction with primers spanning a region of the VE-cadherin promoter containing 4 ETS-binding sites.30 The amplified product of 140 bp (base pair) was enriched in the Erg-precipitated chromatin sample (Erg) compared with the negative control IgG (Neg). To ensure the specificity of Erg binding to the VE-cadherin promoter, PCR amplification was also performed on the same samples using primers specific to the 3′ region of the VE-cadherin locus as negative control. PCR using the negative control primers gave rise to equivalent amounts of product from both samples, indicating nonspecifically captured DNA. (G) An Erg cDNA expression plasmid (pSG5Erg) was cotransfected with the VE-cadherin promoter-luciferase construct in HeLa cells, and luciferase activity was measured. Values are represented as the fold change in relative luciferase activity over the empty pGL4 vector alone. The VE-cadherin promoter was also cotransfected with an empty expression plasmid (pSG5). Erg transactivates the VE-cadherin promoter, **P < .01. Values are means plus or minus SEM, n = 5.
