UC San Diego

Cardiovascular disease (CVD) is a leading cause of deaths in the United States, however the underlying inflammatory pathways that contribute to CVD are poorly understood. Endothelial dysfunction is a hallmark of CVD and induced by various inflammatory mediators, many of which signal through G-protein coupled receptor (GPCRs). Thrombin, an inflammatory mediator, is generated in response to vascular injury and disrupts endothelial barrier integrity and promotes vascular leakage, a hallmark of inflammation. Thrombin activates endothelial protease-activated receptor-1 (PAR1) to disrupt endothelial barrier through the RhoA/myosin light chain (MLC) pathway. However, thrombin/PAR1-stimulated p38 mitogen-activated protein kinase (MAPK) signaling also promotes barrier permeability through a pathway that does not integrate with the RhoA/MLC signaling, suggesting that p38 acts through an uncharacterized pathway to promote inflammatory responses in endothelial cells. Our previous work showed that thrombin-activated PAR1 ubiquitination drives the recruitment of TAB2-TAB1 protein complexes on endosomes to trigger non-canonical p38 activation and endothelial barrier disruption. The mechanisms that regulate PAR1 ubiquitination/de-ubiquitination are not known. This proposal aims to understand the molecular processes that regulate PAR1-ubiquitin-driven p38 signaling and most importantly its impact on endothelial barrier integrity. The central hypothesis is that de-ubiquitination of PAR1 leads to termination of p38-mediated pro-inflammatory signaling. Using advanced biochemical, microscopy and genome-wide RNAi screening methods, we for the first time investigate molecular determinants that regulate the dynamics of PAR1-ubiquitination and the impact on endothelial proinflammatory responses in vitro using human endothelial cells. We reveal new information regarding the function of PAR1 ubiquitination in driving non-canonical endosomal p38 MAPK signaling in endothelial barrier disruption, a hallmark of inflammation, which will provide an opportunity for the development new strategies for the treatment of inflammatory responses associated with CVD.