A key inciting event in atherosclerosis
A key inciting event in atherosclerosis is the deposition of circulating low-density lipoprotein (LDL) in the subendothelial space of major arteries. In the subendothelium, native LDL is no longer protected by circulating antioxidants and is oxidatively modified to oxidized LDL (OxLDL) . OxLDL is no longer recognized by the LDL receptor but rather by scavenger receptors such as CD36 on the surface of the macrophage . Once ligated by OxLDL, CD36 recruits Toll-like receptors (TLR) 4 and 6 and activates an intracellular signaling cascade that requires adapter proteins including tumor necrosis factor receptor associated factors (TRAFs) [8,9]. This signaling cascade phosphorylates and activates the p65 subunit of NF-κB resulting in secretion of pro-inflammatory mediators that perpetuate the inflammatory response and establish the atherosclerotic lesion in the vessel wall . Hence, surface receptor activation coupled to signal transduction through key intermediates that stimulate the cytokine response is critical in mediating actions of modified LDL.
It has been shown that the protein stability of TLRs and signaling intermediates such as TRAF proteins are important in controlling pro-inflammatory responses . Ubiquitination controls the lifespan of the majority of proteins and involves a hierarchical, exquisite system. Ubiquitination involves the stepwise transfer of ubiquitin from an E1 ubiquitin-activating enzyme to an E2 ubiquitin-conjugating enzyme, and finally to an E3 ubiquitin ligase complex . In the final step of the reaction, the E3 ubiquitin ligase transfers ubiquitin chains to the substrate to facilitate degradation by the 26S proteasome or sorting to the endosome-lysosome pathway. Two E1 enzymes, almost 40 E2 enzymes, and >1000 E3 enzymes have been identified in mammalian cholecystokinin receptor . The Skp-Cullin-F-box (SCF) superfamily represents the largest group of E3 ligases that mediate critical roles in cell biology, including tumorigenesis and inflammation. Within the SCF apparatus is a receptor module, termed F-box protein that binds substrates. We previously identified and characterized the molecular behavior of F-box protein FBXO3, that targets another F-box subunit, FBXL2, that in turn controls the stability of TRAF proteins . Therefore, inhibition of FBXO3 increases FBXL2 which decreases levels of TRAF proteins to limit inflammation. TRAF proteins also link cell-surface receptors such as CD36 to NF-κB which appears to be controlled, in part, by the SCFFBXO3 E3 ligase . FBXO3 is a particularly attractive drug target in atherosclerosis because a small molecule inhibitor of FBXO3 is effective in preclinical studies .
In this study, we implicate SCFFBXO3 E3 ligase as a critical modulator of inflammation in atherosclerosis and demonstrate the efficacy of a small molecule FBXO3 inhibitor in suppressing inflammatory responses important in atherosclerosis. Specifically, individuals carrying a hypofunctioning genetic variant of FBXO3 manifest less atherosclerosis. Also, FBXO3 protein levels are increased in atherosclerotic plaques from subjects with symptomatic rather than asymptomatic atherosclerosis. Further, depletion or chemical inhibition of FBXO3 protein abolishes inflammatory responses to OxLDL without altering OxLDL uptake in macrophages. Thus, these studies implicate FBXO3 as a unique, druggable target for anti-inflammatory therapy in atherosclerosis.
Materials and methods
Discussion The role of pro-inflammatory pathways linked to the ubiquitin-proteasome system in the pathogenesis of atherosclerosis is an emerging area of investigation. Indeed, studies demonstrate putative roles of proteasomal blockade, protective effects of ubiquitin E3 ligases, and identification of related susceptibility genes, in the pathobiology of atherosclerosis . This study implicates, for the first time, the SCFFBXO3 E3 ligase as a unique molecular regulator of inflammation in atherosclerosis. Specifically, we observed that i) individuals carrying a hypofunctioning genetic variant of FBXO3 manifest less atherosclerosis, ii) FBXO3 protein is present in cells of monocytic lineage within atherosclerotic plaques and its levels are elevated in subjects with symptomatic disease, and iii) FBXO3 inhibition via genetic silencing or chemical inhibition reduces OxLDL induced pro-inflammatory signal events in macrophages. Although additional proof-of-concept studies will be needed, these data suggest that selective FBXO3 targeting might serve as a potentially attractive pharmacotherapeutic strategy in this cardiovascular disorder.