Endothelial ROBO4 Suppresses PTGS2/COX-2 Expression and Inflammatory Diseases

Endothelial cells (ECs) form a barrier between the vessel lumen and the vessel wall, and due to their ability to affect which factors enter the vessel wall, they have been shown to play a critical role in an array of diseases.  In particular, ECs profoundly affect inflammatory-related diseases, because they can regulate immune cell migration and activity by increasing their vascular permeability and upregulating their inflammatory genes and cytokines. Key targets such as roundabout 4 (ROBO4), an endothelial-specific membrane protein, have been identified to affect EC function in an inflammatory state; however, the precise signaling mechanisms have not been fully elucidated.  Recently, Tanaka et al. identified a novel ROBO4 complex that regulates Rac1 activation to control prostaglandin-endoperoxide synthase 2 (PTGS2) expression and vascular permeability.  Preliminary RNA-Seq studies were performed on control and ROBO4-silenced cells. Of the thousands of genes that were differentially expressed, PTGS2 was identified as a critical target because it appeared most frequently in the top 30 categories of inflammatory-related genes and was clustered in a group of genes that altered EC cell migration.  Follow-up studies determined the mechanism by which ROBO4 regulates PTGS2 and identified a prolonged Rac1 activation as a critical mechanism in their siROBO4/TNF model.   Interestingly, Rac1 activation persisted for hours compared to 30 minutes, and blocking this Rac1 activation with an inhibitor was sufficient to block PTGS2 increases. The group further dissected this signaling axis and found that ROBO4 affects Rac1 activation via a TNF receptor-associated factor 7 (TRAF7) ubiquitin mechanism where ROBO4 functions as a scaffold to bring TRAF7 in proximity with IQ motif-containing GTPase-activating protein 1 (IQGAP1), which leads to ubiquitination of IQGAP1. Ubiquitination of IQGAP1 disrupted the interaction between IQGAP1 and Rac1 and led to Rac1 inactivation.  In siROB4 cells, the IQGAP1 and Rac1 interaction was not disrupted by this mechanism, which led to the prolonged activation of Rac1, JNK downstream activation, and an upregulation of PTGS2 and enhanced hyperpermeability. Interestingly, the group performed a collegen-induced arthritis model in control and ROBO4 null mice, and found that the arthritis phenotypes were exacerbated in mice that lacked ROBO4. Cytoskeleton’s Rac1 G-LISA Activation Assay (Cat. # BK128) was used to measure active Rac1 in response to TNF in ROBO4-depleted cells and was a critical tool in identifying important changes in Rac1 in this study.