RhoB controls endothelial barrier recovery by inhibiting Rac1 trafficking to the cell border

Endothelial barrier dysfunction underlies chronic inflammatory diseases. In searching for new proteins essential to the human endothelial inflammatory response, we have found that the endosomal GTPase RhoB is up-regulated in response to inflammatory cytokines and expressed in the endothelium of some chronically inflamed tissues. We show that although RhoB and the related RhoA and RhoC play additive and redundant roles in various aspects of endothelial barrier function, RhoB specifically inhibits barrier restoration after acute cell contraction by preventing plasma membrane extension. During barrier restoration, RhoB trafficking is induced between vesicles containing RhoB nanoclusters and plasma membrane protrusions. The Rho GTPase Racl controls membrane spreading and stabilizes endothelial barriers. We show that RhoB colocalizes with Racl in endosomes and inhibits Racl activity and trafficking to the cell border during barrier recovery. Inhibition of endosomal trafficking impairs barrier reformation, whereas induction of Racl translocation to the plasma membrane accelerates it. Therefore, RhoB-specific regulation of Racl trafficking controls endothelial barrier integrity during inflammation.