CSN5 inhibition triggers inflammatory signaling and Rho/ROCK-dependent loss of endothelial integrity

Abstract

RhoGTPases regulate cytoskeletal dynamics, migration and cell-cell adhesion in endothelial cells. Besides regulation at the level of guanine nucleotide binding, they also undergo post-translational modifications, for example ubiquitination. RhoGTPases are ubiquitinated by Cullin RING ligases which are in turn regulated by neddylation. Previously we showed that inhibition of Cullin RING ligase activity by the neddylation inhibitor MLN4924 is detrimental for endothelial barrier function, due to accumulation of RhoB and the consequent induction of contractility. Here we analyzed the effect of pharmacological activation of Cullin RING ligases on endothelial barrier integrity in vitro and in vivo . CSN5i-3 induced endothelial barrier disruption and increased macromolecule leakage in vitro and in vivo . Mechanistically, CSN5i-3 strongly induced the expression and activation of RhoB and to lesser extent of RhoA in endothelial cells, which enhanced cell contraction. Elevated expression of RhoGTPases was a consequence of activation of the NF-κB pathway. In line with this notion, CSN5i-3 treatment decreased IκBα expression and increased NF-κB-mediated ICAM-1 expression and consequent adhesion of neutrophils to endothelial cells. This study shows that sustained neddylation of Cullin RING-ligases leads to activation the NF-κB pathway in endothelial cells, elevated expression of RhoGTPases, Rho/ROCK-dependent activation of MLC and disruption of the endothelial barrier.

COP9 signalosome is an essential and druggable parasite target that regulates protein degradation

“…2B, fig. S3E), which can sometimes run lower than its predicted molecular weight (25). Therefore, we tested if cullin1 neddylation is altered upon CSN5 knockdown, and found that knockdown cells accumulated neddylated cullin1 by immunoblot analysis using antibodies specific for E. histolytica cullin1 and Nedd8 (Fig.…”

COP9 signalosome is an essential and druggable parasite target that regulates protein degradation

“…histolytica cullin1 protein (EHI_118180) [ 32 ] which has the conserved neddylation site ( S4 Fig ). Immunoblot analysis revealed the most intense neddylated band after CSN5 disruption being above the 75 KDa mark, which includes neddylated cullin1 ( Fig 2B , S3E Fig and S6D Fig ), which can sometimes run lower than its predicted molecular weight [ 48 ]. Therefore, we tested if cullin1 neddylation is altered upon CSN5 knockdown, and found that knockdown cells accumulated neddylated cullin1 by immunoblot analysis using antibodies specific for E .…”

The pivotal roles of neddylation pathway in immunoregulation

“…The state-of-art research has proven that treatment of human umbilical vein endothelial cells with a COP9 signalosome inhibitor (pharmacological activation of CRLs) enhanced intercellular cell adhesion molecule (ICAM) expression and resulted in consequent adhesion of neutrophils to endothelial cells. 55 Moreover, the glioma cells after MLN4924 treatment were reported to have a stronger potential to induce T-cell anergy. 56 Recently, our colleagues uncovered that neddylation inactivation in tumor cell increased C-C motif chemokine ligand 2 (CCL2) secretion and then promoted tumor-associated macrophage infiltration.…”

“…In a consequence, typical targets for NF-κB such as proinflammatory cytokines (TNF-α, IL-6), chemokines (CCL2/MCP1), antirival factor (IFN-β) and adhesion molecules (ICAM) are downregulated. 25,55,57,65 However, what's puzzling is that neddylation inactivation usually promotes the accumulation of phosphorylated IκBα, rather than total IκBα, in some studies. 34,66 It may be interpreted that other protein degradation pathways are involved in the regulation.…”

“…Neddylation is also considered to regulate substrate proteins in other types of cells such as endothelial cells or epithelial cells, and their interaction with immune cells is influenced as a consequence. The state‐of‐art research has proven that treatment of human umbilical vein endothelial cells with a COP9 signalosome inhibitor (pharmacological activation of CRLs) enhanced intercellular cell adhesion molecule (ICAM) expression and resulted in consequent adhesion of neutrophils to endothelial cells 55 . Moreover, the glioma cells after MLN4924 treatment were reported to have a stronger potential to induce T‐cell anergy 56 .…”