Vascular endothelial (VE)-cadherin, a major endothelial adhesion molecule, 
regulates vascular permeability, and increased vascular permeability has been 
observed in several cancers. The aim of this study was to elucidate the role of 
the NEDD8-Cullin E3 ligase, in maintaining barrier permeability. To this end, we 
investigated the effects of the inhibition of Cullin E3 ligases, by using 
inhibitors and knockdown techniques in HUVECs. Furthermore, we analyzed the mRNA 
and protein levels of the ligases by quantitative RT-PCR and Western blotting, 
respectively. The results revealed that NEDD8-conjugated Cullin 3 is required 
for VE-cadherin-mediated endothelial barrier functions. Treatment of HUVECs with 
MLN4924, a chemical inhibitor of the NEDD8-activating enzyme, led to high 
vascular permeability due to impaired cell-cell contact. Similar results were 
obtained when HUVECs were treated with siRNA directed against Cullin 3, one of 
the target substrates of NEDD8. Immunocytochemical staining showed that both 
treatments equally depleted VE-cadherin protein localized at the cell-cell 
borders. However, quantitative RT-PCR showed that there was no significant 
difference in the VE-cadherin mRNA levels between the treatment and control 
groups. In addition, cycloheximide chase assay revealed that the half-life of 
VE-cadherin protein was dramatically reduced by Cullin 3 depletion. Together, 
these findings suggest that neddylated Cullin 3 plays a crucial role in 
endothelial cell barrier function by regulating VE-cadherin.