Role of the COP9 signalosome and IKK complexes in atherosclerosis

The COP9 signalosome (CSN), a multi-functional protein complex involved in the regulation of E3 ubiquitin ligases, has been described as a regulator of NF-kappaB signaling, a function assigned to the metalloisopeptidase activity of its subunit CSN5. However, its effect on NF-kappaB activation and pro-atherogenic responses in endothelial cells and macrophages and in atherogenesis in vivo remains unknown. This study sought to identify a role of the CSN complex and in particular CSN5 in IKK/NF-kappaB signaling in atherogenic endothelial cells in vitro and in murine atherosclerosis in vivo. Endothelial cells (ECs) are not just barrier bystanders, but actually determine whether immune cells will work within the limits of homeostasis or go overboard into autoimmunity and inflammation. As the vascular endothelium plays a pivotal role in the inflammatory reactions driving atherosclerosis, the first part of this study focused on the role of CSN5 in modulating inflammatory and atherogenic processes in ECs through the regulation of NF-kappaB. By using ECs, this study for the first time demonstrates endothelial CSN5 to reduce inflammatory responses and mononuclear cell arrest by impairing NF-kappaB activation, proposing that CSN5 confers atheroprotective effects in the endothelium. This was mechanistically shown by co-immunoprecipitation in human umbilical vein endothelial cells (HUVECs) that revealed the presence of a supercomplex between IKK and the CSN, which dissociates upon TNFalpha administration. The CSN was found to attenuate the phosphorylation of IKK when cells were treated with siRNAs targeting CSN subunits 2 and 5. Furthermore, transient overexpression of CSN5 protected IkappaB-alpha from degradation and reduced NF-kappaB activation upon TNFalpha stimulation, whereas silencing RNA-mediated CSN5 depletion enhanced TNFalpha-induced IkappaB-alpha degradation and the concomitant NF-kappaB activity in NF-kappaB binding and luciferase reporter assays. Sequentially, NF-kappaB-dependent expression of chemokines and adhesion molecules was upregulated, as measured by real-time PCR and flow cytometry, which translated into an increased arrest of perfused monocytes on TNFalpha-stimulated, CSN5-depleted HUVECs. Importantly, double-immunostaining for CSN and CD31 confirmed the expression of CSN subunits in the endothelial layer of human atherosclerotic lesions, and revealed an increased expression of CSN5 upon atheroprogression. The second part of this work intended to elucidate the functional role of CSN5 in murine atherosclerosis in vivo. Macrophages are key cells not only in acute inflammation but also in chronic inflammation as they release a number of cytokines and other factors upon activation. Chronic inflammatory processes underlie atherosclerosis and macrophages have been shown to be key cells in atherosclerotic processes. Employing a macrophage-specific deletion of Csn5 in a murine atherosclerosis model, this study identifies Csn5 as a novel player in atherosclerosis. Ablation of Csn5 was found to exacerbate murine atherosclerosis, an observation that confirms the atheroprotective role of CSN5 demonstrated for the first time in this study. The evidence presented in this thesis sets the pace to pursue the functional role of CSN5 and the entire CSN complex in atherosclerosis in a broader manner including EC- and SMC-specific knockout of Csn5 in vivo by conditional deletion. This could also be extended to other Csn subunits and the various proatherogenic mouse models.