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Endothelial dysfunction is induced by inflammatory mediators that signal through GPCRs and a hallmark of inflammation. Protease‐activated receptor‐1 (PAR1), a GPCR for thrombin, induces endothelial dysfunction via multiple signal transduction pathways including p38 mitogen‐activated protein kinase (MAPK). We showed that thrombin‐stimulates K63‐linked ubiquitination of PAR1 that drives recruitment of transforming growth factor‐β‐activated kinase‐1 binding protein‐2 (TAB2), an adaptor protein that binds TAB1, which triggers p38 activation on endosomes and promotes endothelial barrier disruption. However, the regulatory processes that control thrombin‐stimulated PAR1 ubiquitin‐dependent p38 inflammatory signaling are not known. The objective of this study was to identify specific deubiquitinases (DUBs) that counter thrombin‐induced ubiquitin‐mediated p38 signaling by conducting an unbiased genome‐wide siRNA library screen targeting all 96 human DUB genes in human cultured endothelial cells. We hypothesize that a PAR1‐specific DUB is essential for controlling proper dynamics of thrombin‐mediated p38 signaling and might be altered in endothelial dysfunction. To systematically identify DUBs, endothelial cells were transfected with pools of 4 siRNAs targeting each of the 96 DUBs and thrombin‐stimulated p38 phosphorylation was assessed by immunoblotting. In parallel, a genome‐wide DUB siRNA library screen was conducted in HeLa cells. We identified 8 different DUBs that preferentially cleave K63‐linked ubiquitin and regulate thrombin‐p38 signaling in both endothelial and HeLa cells. A time‐course analysis of thrombin‐stimulated p38 signaling confirmed that depletion of each of the 8 DUBs alone caused an increase in the magnitude and/or duration of p38 signaling. Among the candidate DUBs, USP34, USP47 and CYLD exhibited the highest expression in both endothelial and HeLa cells detected by RT‐qPCR and were further examined. Using individual siRNAs targeting each of the 3 candidate DUBs, we validated that knockdown of either USP34, USP47 or CYLD alone was sufficient to markedly enhance thrombin‐induced p38 signaling in endothelial cells. New data examining the function of CYLD on thrombin‐induced inflammatory responses will be presented. While ubiquitin‐dependent signaling is widespread, our knowledge of the processes that control ubiquitin‐mediated GPCR signaling is limited, especially related to the function of GPCR‐specific DUBs. In this study, we report the first genome‐wide DUB siRNA library screen on GPCR signaling and identified a subset of DUBs that specifically regulate PAR1‐p38 inflammatory signaling that may serve as important therapeutic targets for vascular inflammation.

Endothelial dysfunction is a hallmark of vascular disease and induced by various inflammatory mediators which signal through GPCRs pathways that are poorly understood. Thrombin induces endothelial inflammatory responses through the activation of protease‐activated receptor 1 (PAR1), a GPCR. Previously, we showed that thrombin‐activated PAR1 is modified by ubiquitin and drives recruitment of transforming growth factor‐β–activated kinase‐1–binding protein 2 (TAB2), an adaptor protein that binds TAB1 to trigger p38‐dependent endothelial barrier disruption, a hallmark of inflammation. However, the regulatory processes that control PAR1‐deubiquitination and associated p38 inflammatory signaling are not known. To fill this gap in knowledge, we sought to identify PAR1‐specific deubiquitinases (DUBs) that removes K63‐linked ubiquitin by conducting an unbiased comprehensive genome‐wide siRNA library screen targeting all 96 human DUB genes in human cultured endothelial cells using multiple PAR1 ubiquitin‐dependent functional readouts. We hypothesize that a PAR1‐specific DUB is essential for terminating thrombin‐induced p38 inflammatory signaling and might be altered in endothelial dysfunction. In an initial screen, endothelial cells were transfected with siRNA pools targeting specific DUBs and PAR1‐ubiquitin dependent responses were assessed. We specifically examined PAR1 cell surface expression, controlled by basal PAR1 ubiquitination, and thrombin‐stimulated p38 activation and downstream inflammatory signaling, which is dependent on agonist‐triggered PAR1 ubiquitination. In preliminary studies have identified several candidates PAR1‐specific DUBs. Currently, we are now investigating the function of top candidate DUBs in regulating the status of PAR1 ubiquitination, cell surface expression and p38 inflammatory signaling. The molecular mechanism responsible for PAR1 deubiquination and relation to controlling the spatio‐temporal dynamics of pro‐inflammatory signaling will be further investigated. Taken together, we anticipate that these studies using a comprehensive siRNA library screening approach will reveal the identity of several DUBS that have important functions in regulating PAR1‐induced ubiquitin driven p38 MAPK endothelial inflammatory signaling. Support or Funding Information Authors acknowledge NIH/NIGMS funding support.