Abstract 5487: The Ubiquitin Ligase Nedd4 Mediates Oxidized Low-Density Lipoprotein Induced Downregulation of Insulin-Like Growth Factor-1 Receptor in Human Aortic Smooth Muscle Cells: Implications for Atherosclerotic Plaque Destabilization
Oxidized low-density lipoprotein (oxLDL) is a modified LDL with major proatherogenic properties, including induction of smooth muscle cell (SMC) apoptosis, which contributes to plaque destabilization. We have previously shown that oxLDL downregulates insulin-like growth factor-1 receptor (IGF-1R) in human SMC and that this is a critical pathway whereby oxLDL triggers SMC apoptosis. In human atherosclerotic plaque, oxLDL co-localizes with areas of SMC IGF-1R downregulation and increased apoptotic rates. To identify the mechanism of oxLDL downregulation of IGF-1R, we exposed SMC to 60 μg/ml oxLDL or native LDL (nLDL) and assessed IGF-1R mRNA level, IGF-1R protein synthesis rate, and IGF-1R protein stability. Although oxLDL decreased IGF-1R mRNA levels by 30 %, IGF-1R protein synthesis rate was not altered. Pulse-chase labeling experiments revealed that oxLDL decreased IGF-1R protein half-life from 24.4 ± 4.7 hours (with nLDL as control) to 12.2 ± 1.7 hours, indicating that the primary mechanism for oxLDL downregulation of IGF-1R is protein degradation. OxLDL induced IGF-1R degradation was accompanied by enhanced receptor ubiquitination, as detected by Western blotting of ubiquitin-conjugated protein immunoprecipitated with anti-IGF-1R antibody. The ubiquitin ligase Nedd4 has recently been identified as important for ligand-induced IGF-1R downregulation. Electroporation of an expression plasmid for a dominant negative form of Nedd4 (transfection efficiency ~60%) markedly inhibited oxLDL downregulation of IGF-1R by 55%. To characterize the oxLDL induced IGF-1R degradation pathway, we tested various protease inhibitors (MG-132, lactacystin, calpain inhibitor III, PD150606, calpeptin, EST, and ALLN) and found that, surprisingly, proteasome selective inhibitors (MG-132, lactacystin) did not block oxLDL induced downregulation of IGF-1R. Only ALLN effectively prevented oxLDL induced downregulation of IGF-1R, consistent with a model in which Nedd4-mediated ubiquitination directs IGF-1R to degradation via a lysosomal cathepsin dependent pathway. Our findings provide novel insights into oxLDL-triggered oxidant signaling and mechanisms of SMC depletion that contribute to plaque destabilization and coronary events.