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 TNFα 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.