12/15-lipoxygenase activity increases the degradation of macrophage ATP-binding cassette transporter G1

Objective - The purpose of this study was to evaluate the effect of 12/15-lipoxygenase (12/15LO) in macrophage ABCG1 expression and function associated with cholesterol efflux. Methods and Results - 12/15LO was stably overexpressed in J774 macrophages. 12/15LO-overexpressing macrophages had a 30% reduction in HDL-mediated cholesterol efflux, corresponding with significantly reduced ABCG1 protein expression. Treatment of 12/15LO-overexpressing macrophages with a 12/15LO ribozyme to reduce 12/15LO restored HDL-mediated efflux and ABCG1 protein expression. Treating macrophages with 12/15LO unsaturated fatty acid substrates or eicosanoid products also reduced HDL-mediated cholesterol efflux. Additionally, both 12/15LO overexpression in macrophages and incubation of macrophages with eicosanoids reduced ABCG1 protein, but not mRNA, expression. However, incubation of macrophages with linoleic or arachidonic acids significantly reduced both ABCG1 mRNA and protein expression, suggesting that 12/15LO substrates and eicosanoid products differentially regulate ABCG1 expression. 12/15LO fatty acids did not decrease ABCG1 translation; however, 12/15LO fatty acids increased ABCG1 degradation when blocked by cyclohexidmide. ABCG1 degradation may be regulated through posttranslational modifications. Treatment with the 12/15LO eicosanoid product 12SHETE increased serine phosphorylation of ABCG1. Conclusions - We conclude that serine phosphorylation may increase the degradation rate of ABCG1, and as a result cause macrophage cholesterol accumulation. These findings provide evidence that 12/15LO activity in the vessel wall contributes to atherogenesis by impairing the macrophage ABCG1 cholesterol efflux pathway.