A functional siRNA screen identifies RhoGTPase-associated genes involved in thrombin-induced endothelial permeability

Abstract

Thrombin and other inflammatory mediators may induce vascular permeability through the disruption of adherens junctions between adjacent endothelial cells. If uncontrolled, hyperpermeability leads to an impaired barrier, fluid leakage and edema, which can contribute to multi-organ failure and death. RhoGTPases control cytoskeletal dynamics, adhesion and migration and are known regulators of endothelial integrity. Knowledge of the precise role of each RhoGTPase, and their associated regulatory and effector genes, in endothelial integrity is incomplete. Using a combination of a RNAi screen with electrical impedance measurements, we quantified the effect of individually silencing 270 Rho-associated genes on the barrier function of thrombin-activated, primary endothelial cells. Known and novel RhoGTPase-associated regulators that modulate the response to thrombin were identified (RTKN, TIAM2, MLC1, ARPC1B, SEPT2, SLC9A3R1, RACGAP1, RAPGEF2, RHOD, PREX1, ARHGEF7, PLXNB2, ARHGAP45, SRGAP2, ARHGEF5). In conclusion, with this siRNA screen, we confirmed the roles of known regulators of endothelial integrity but also identified new, potential key players in thrombin-induced endothelial signaling.

High Throughput strategies Aimed at Closing the GAP in Our Knowledge of Rho GTPase Signaling

“…Screening approaches in mammalian cells were also useful to probe the function of Rho GTPases in physiological processes. Notably, Rho GTPases network components involved in thrombin-induced endothelial permeability in primary umbilical vein endothelial cells were identified using a siRNA screen that functionally tested 270 human Rho GTPases and Rho-associated genes [ 88 ]. A total of 15 top hits that modify the response to thrombin were identified [ 88 ].…”

“…Notably, Rho GTPases network components involved in thrombin-induced endothelial permeability in primary umbilical vein endothelial cells were identified using a siRNA screen that functionally tested 270 human Rho GTPases and Rho-associated genes [ 88 ]. A total of 15 top hits that modify the response to thrombin were identified [ 88 ]. Intriguingly, the depletion of the Rho GTPase RHOD led to the most potent disruption of endothelial barrier integrity, which revealed its important role in this process [ 88 ].…”

“…A total of 15 top hits that modify the response to thrombin were identified [ 88 ]. Intriguingly, the depletion of the Rho GTPase RHOD led to the most potent disruption of endothelial barrier integrity, which revealed its important role in this process [ 88 ]. In another study, Zaritsky et al rather designed a shRNA screen targeting 80 RhoGEFs in human bronchial epithelial cell monolayers to identify the ones that regulate the long-range collective migration in these cells [ 89 ].…”

Nanoplasmonics-enhanced label-free imaging of endothelial cell monolayer integrity

“…Cell responses following protease-activated receptor 1 (PAR1) activation by thrombin have been studied for its role in haemostasis and inflammation mechanisms (Coughlin, 2000). Thrombin has been extensively used to destabilize endothelial cell layers for instance to investigate its effect on barrier function permeability (Amado-Azevedo et al, 2018;Bae et al, 2009;Minami et al, 2004;O'Brien et al, 2000;Söllradl et al, 2018a;Troyanovsky et al, 2008;van der Heijden et al, 2011). Thrombin binds with the cell protease-activated receptor 1 (PAR1), activates intracellular signaling cascades which induces endothelial cell contraction, cell-cell molecular junction disassembly and the modulation of cell attachment onto the substrate (Opal and van der Poll, 2015).…”

An RNAi screen unravels the complexities of Rho GTPase networks in skin morphogenesis

“…In mammals, there are 20 Rho GTPases and nearly 150 regulators. Cell culture screening approaches have uncovered roles played by Rho GTPases and their regulators in cellular processes (Duquette and Lamarche-Vane, 2014; Amado-Azevedo et al, 2018; Tajadura-Ortega et al, 2018; Pascual-Vargas et al, 2017). Thus far however, in vivo analyses of Rho GTPase functions in morphogenetic processes have been largely restricted to signaling by the three prototypical members RAC, RHO and CDC42 (Hodge and Ridley, 2016; Heasman and Ridley, 2008; Van Aelst and Symons, 2002; Duquette and Lamarche-Vane, 2014).…”