Identification of Deubiquitinases (DUBs) that modulate PAR1‐mediated p38 MAPK inflammatory signaling in endothelial cells

Abstract

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. To address this gap in knowledge, we sought 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. Currently, I am examining the impact of USP34‐mediated modulation of thrombin‐p38 signaling on endothelial inflammatory responses. I will present new data examining the role of USP34 on thrombin‐induced endothelial barrier disruption and induction of cytokine expression. 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. Here, 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.