Endothelial ROBO4 suppresses PTGS2/COX-2 expression and inflammatory diseases

Endothelial cells control inflammation in inflammatory and infectious diseases through regulating endothelial gene expression and permeability. Roundabout4 (Robo4) is an endothelial-specific protein that stabilizes endothelial cells. Robo4 has been shown to ameliorate mouse inflammatory and severe infectious diseases, such as sepsis and COVID-19 by reducing vascular permeability. Despite this, it remains uncertain whether these are the only mechanisms by which Robo4 ameliorates the diseases. In this study, we carried out an RNA-seq analysis to investigate the genes regulated by Robo4 in endothelial cells stimulated by TNFa and identified gene, prostaglandin-endoperoxide synthase 2 (PTGS2) , which codes for Cycloocygenase-2 (COX-2). Mechanistic analysis revealed that Robo4 curbs COX-2 expression and endothelial hyperpermeability by preventing prolonged Rac1 activation. Analysis of Robo4 interacting protein identified IQ motif containing GTPase activating protein 1 (IQGAP1) that maintains active Rac1. Robo4 enhanced ubiquitination of IQGAP1 with a ubiquitin E3 ligase, TNF receptor-associated factor 7 (TRAF7), to inactivate Rac1. Finally, Robo4 deficiency exacerbates COX-2 -associated inflammatory diseases, including arthritis, edema, and pain in mouse models. Taken together, Robo4 inhibits Rac1 activation by interacting with TRAF7 and IQGAP1, thereby suppressing the endothelial expression of COX-2 and reducing hyperpermeability. Thus, we uncovered further Robo4 capabilities that suppresses COX-2 and inflammatory diseases, as well as their underlying mechanisms, indicating that endothelial Robo4 is a potential therapeutic target for diverse inflammatory diseases. Overall design: The Robo4 siRNA-transfected or TNFa-treated HUVECs were collected, and then total RNA was collected and RNA-seq was performed.内皮细胞（endothelial cells）通过调控内皮基因表达与血管通透性，参与炎症及感染性疾病中的炎症调控过程。Roundabout4（Robo4）是一种内皮特异性蛋白，可维持内皮细胞的稳定性。已有研究证实，Robo4可通过降低血管通透性，缓解小鼠的炎症反应与脓毒症、新型冠状病毒肺炎（COVID-19）等重症感染性疾病。但目前尚不明确上述是否为Robo4改善疾病的全部机制。 本研究通过RNA测序（RNA-seq）分析，探究了肿瘤坏死因子α（TNF-α）刺激的内皮细胞中Robo4所调控的基因，并鉴定出前列腺素内过氧化物合酶2（prostaglandin-endoperoxide synthase 2，PTGS2）——其编码产物为环氧合酶2（Cyclooxygenase-2，COX-2）。机制研究显示，Robo4可通过抑制Rac1的过度激活，下调COX-2的表达并改善内皮细胞高通透性。 对Robo4互作蛋白的分析发现，含IQ基序的GTP酶激活蛋白1（IQ motif containing GTPase activating protein 1，IQGAP1）可维持Rac1的激活状态。Robo4联合泛素E3连接酶肿瘤坏死因子受体相关因子7（TNF receptor-associated factor 7，TRAF7），可促进IQGAP1的泛素化，进而使Rac1失活。 最终实验表明，在小鼠模型中，Robo4缺陷会加剧COX-2相关的炎症性疾病，包括关节炎、水肿与疼痛。综上，Robo4可通过与TRAF7及IQGAP1相互作用，抑制Rac1的激活，进而抑制内皮细胞中COX-2的表达并降低血管高通透性。 本研究揭示了Robo4抑制COX-2表达与炎症性疾病的全新功能及其潜在分子机制，表明内皮细胞中的Robo4可作为多种炎症性疾病的潜在治疗靶点。 实验设计：收集转染Robo4小干扰RNA（siRNA）或经TNF-α处理的人脐静脉内皮细胞（Human Umbilical Vein Endothelial Cells，HUVECs），提取总RNA并开展RNA-seq测序。