iNOS Activation Regulates β-catenin Association with Its Partners in Endothelial Cells

Abstract

BackgroundSignals that disrupt β-catenin association to cadherins may influence the translocation of β-catenin to the nucleus to regulate transcription. Post-translational modification of proteins is a signalling event that may lead to changes in structural conformation, association or function of the target proteins. NO and its derivatives induce nitration of proteins during inflammation. It has been described that animals treated with NO donors showed increased permeability due to modulation of VE-cadherin/catenin complex. We, therefore, aim to evaluate the effect of iNOS activation on the expression, nuclear localisation and function of β-catenin in endothelial cells.Methodology/Principal FindingsExpression, nuclear localisation, post-translational modifications and function of β-catenin was analysed by cell fractionation, immunoprecipitation, immunoblots, QRT-PCR and permeability assays in murine endothelial cells (H5V). Influence of macrophage activation on expression of VE-cadherin/p120-catenin/β-catenin complex in co-cultured H5V cells was also assessed. Activation of macrophages to produce NO provoked a decrease in VE-cadherin/p120-catenin/β-catenin expression in H5V cells. Phosphorylation of β-catenin, p120-catenin and VE-cadherin, and reduction in the barrier properties of the cell monolayer was associated with iNOS induction. Moreover, high NO levels provoked nitration of β-catenin, and induced its translocation to the nucleus. In the nucleus of NOS activated cells, nitration levels of β-catenin influenced its association with TCF4 and p65 proteins. High levels of NO altered β-catenin mediated gene expression of NFκB and Wnt target genes without affecting cell viability.ConclusionsNOS activity modulates β-catenin post-translational modifications, function and its association with different partners to promote endothelial cell survival. Therapeutic manipulation of iNOS levels may remove a critical cytoprotective mechanism of importance in tumour angiogenesis.

Effect of nitric oxide synthase on multiple drug resistance is related to Wnt signaling in non-small cell lung cancer

“…In inflammation conditions, the iNOS gene is often activated, resulting in the production of NO. Thus, the pro-inflammatory cytokines TNF-α, IL-1β and IFN-γ were used to trigger the expression of iNOS in our experiment (14)(15)(16)(17). To investigate the Wnt signaling pathway, DKK-1 was added into the medium to inhibit the Wnt pathway.…”

Protein Interactions at Endothelial Junctions and Signaling Mechanisms Regulating Endothelial Permeability

“…Nitration of junctional proteins, such as p190RhoGAP, which is associated with p120-catenin 157 , and β-catenin itself 219 , facilitates disassembly of the VE-cadherin adhesion complex. The pro-inflammatory mediator serine protease α-thrombin triggers nitration of p190RhoGAP on Tyr1105 downstream of eNOS-mediated NO redox signaling 157 .…”

Control of vascular permeability by adhesion molecules

“…Others, such as VEGF, can increase nuclear b¡catenin activity by stimulating nitrosylation of b¡catenin. 126 Nitrosylation of b¡catenin by VEGF-induced eNOS, or b¡catenin nitration by macrophage derived NO, 127 leads to the shuttling of b¡catenin into the nucleus. There, it can activate transcription by binding to the TCF/LEF family of transcriptional repressors.…”

“…Interestingly b¡catenin nitration appears to also promote the interaction of nuclear b¡catenin with NFkB p65. 127 However, b¡catenin nuclear localization following loss of VE-cadherin adhesion does not necessarily require inactivation of the degradation complex or additional post-translational modification of b¡catenin. Taddei, et al showed that VE-cadherin null cells exhibit higher levels of nuclear b¡catenin.…”