GSE161021
TNF-induced ArhGEF10 activates RhoB in human endothelial cells and contributes to tight junction disruption and barrier breakdown
Rationale: Endothelial cells (ECs) line the inner surface of continuous capillaries and form a permselective barrier between blood and tissues to limit paracellular fluid and solute flux. The capillary barrier is maintained by intercellular tight junctions (TJs) which are regulated, in part, by differential activity of small GTPases. Tumor necrosis factor (TNF) causes TJ disassembly in a process dependent upon NF-?B-mediated gene expression. Activated (GTP-bound) RhoB has been identified as a downstream effector of TNF-induced leak both through Rho-associated coiled-coil kinase-mediated phosphorylation of RelA, enhancing TNF-induced NF-?B-mediated gene activation, and through sequestration of Rac1 away from the cell junction, preventing barrier restoration. To date, no TNF-induced, RhoB-selective GTP exchange factor (GEF) has been identified in ECs.   Objective: Identify a TNF-induced RhoB specific GEF in capillary ECs.  Method and Results: human dermal microvascular ECs (HDMECs) were used because they form TJs. Candidate GEFs were identified by whole transcriptome profiling of HDMECs with or without TNF stimulation. Fifty-seven of 73 known GEFs were expressed in HDMECs and 17 were significantly upregulated by TNF. Functional impact of each of the latter group was assessed by measuring the effects of depletion by short interfering RNA knockdown on TNF-induced reduction of trans-endothelial electrical resistance (TEER) of HDMEC monolayers. Depletions of ArhGEF10 or RhoB or double depletion of ArhGEF10/RhoB similarly decreased TNF-mediated reduction of TEER. ArhGEF10 knockdown prevented TNF-induced disruption of TJ proteins assessed by confocal microscopy. TNF-induced increases in the levels of activated RhoB in HDMEC lysates were markedly reduced with only minimal alterations in RhoA or RhoC activation. Finally, immunoisolated ArhGEF10 selectively catalyzed GTP exchange for GDP in RhoB, but not RhoA or RhoC, in vitro.   Conclusion: ArhGEF10 is a TNF-induced RhoB-selective GEF for RhoB in cultured human capillary ECs that contributes to TJ disruption. Overall design: Whole transcriptome analysis of cultured HDMECs, with and without treatment with TNF with 3 replicates per condition