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

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. Overall design: Whole transcriptome analysis of cultured human umbilical vein endothelial cells (HUVEC) with and without treatment with TNF with 3 replicates per condition

在急性危重症中，毛细血管屏障可发生破坏，进而引发低血容量、细胞外水肿、组织缺氧甚至死亡。依赖紧密连接的毛细血管屏障的修复过程由小GTP酶（small GTPases）调控，但目前对该病理情境下发挥关键作用的特异性调控分子尚未得到充分阐释。对形成紧密连接的人皮肤微血管内皮细胞（human dermal microvascular ECs, HDMECs）与形成黏着连接的人脐静脉内皮细胞（human umbilical vein EC, HUVECs）开展转录组分析后发现，肿瘤坏死因子（TNF）刺激前后，ARHGEF12在HDMECs中存在显著差异表达调控。敲低ArhGEF12的HDMECs会出现更为显著的TNF诱导的跨内皮电阻（trans-endothelial electrical resistance）下降，以及紧密连接蛋白claudin-5与ZO-1的结构破坏。ArhGEF12属于RhoA鸟苷酸交换因子（RhoA-GEF），这一学界公认的结论与我们的实验观测结果并不一致。对敲低ArhGEF12并经TNF处理的HDMECs进行下拉激活实验后发现，其Rap1A活性在4小时后降低，而12小时后RhoA活性出现反常升高。在无细胞体系的下拉激活实验中，免疫沉淀得到的ArhGEF12可有效激活Rap1A与RhoA，但无法激活Rap2A-C、RhoB-C，乃至与Rap1A序列同源性高达95%的Rap1B。本研究结论表明：在形成紧密连接的内皮细胞中，ArhGEF12可选择性激活Rap1A，通过独立于经典cAMP介导的Epac1激活通路的机制，促进毛细血管屏障的修复。ArhGEF12对RhoA、Rap1A而非Rap1B展现出独特的体外双重底物特异性，这一现象无法通过序列同源性解释，有待进一步研究。实验整体设计：对经TNF处理与未处理的培养人脐静脉内皮细胞（HUVECs）开展全转录组分析，每组设置3次生物学重复。