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ARHGEF12 activates Rap1A and not RhoA in human capillary endothelial cells to reduce tumor necrosis factor-induced leak

ABSTRACT: In acute critical illness the capillary barrier may break down resulting in hypovolemia, extracellular edema, tissue dysoxia and even death. Restoration of the tight junction dependent capillary barrier is regulated by small GTPases, the specific regulatory molecules most active in this setting are not well described. Transcriptional profiling of tight junction forming human dermal microvascular ECs (HDMECs) and adherens junctions forming human umbilical vein EC (HUVECs) demonstrate ARHGEF12 is significantly differentially regulated in HDMECs before and after stimulation with tumor necrosis factor (TNF). HDMEC depleted of ArhGEF12 demonstrate significantly exacerbated TNF-induced decrease in trans-endothelial electrical resistance and disruption of tight junction proteins claudin-5 and ZO-1. ArhGEF12 is a RhoA-GEF, an established finding which is inconsistent with our observed results. Pulldown activation assays from of HDMECs depleted of ArhGEF12 and treated with TNF show decreased Rap1A activity after four hours and paradoxically increased RhoA activity after 12 hours. In cell-free pulldown assays, immunoprecipitated ArhGEF12 effectively activates Rap1A and RhoA and not Rap2A-C, RhoB-C or even Rap1B which shares 95% sequence identity with Rap1A. We conclude that in tight junction forming EC, ArhGEF12 selectively activates Rap1A to promote capillary barrier restoration in a mechanism independent of traditionally described cAMP-mediated Epac1 activation. The unique dual in vitro specificity of ArhGEF12 for RhoA, Rap1A and not Rap1B cannot be explained by sequence identity and requires additional investigation.

ORGANISM(S): Homo sapiens

PROVIDER: GSE190181 | GEO | 2021/12/07

REPOSITORIES: GEO

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Project description: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-κB-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-κB-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.

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ARHGEF12 activates Rap1A and not RhoA in human capillary endothelial cells to reduce tumor necrosis factor-induced leak

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