Abstract 826: 12/15-Lipoxygenase Activity Inhibits Cholesterol Efflux from Macrophages by Enhancing Degradation of ATP-Binding Cassette Transporter G1
The enzyme 12/15-lipoxygenase (12/15LO) is a nonheme iron-containing dioxygenase that incorporates molecular oxygen in a stereospecific manner into arachidonic and linoleic acids to form 12- and 15-S-hydroxyeicosatetraenoic acids (12SHETE/15SHETE) and 13-S-hydroxyoctadecadienoic acid (13SHODE) respectively. Disruption of the 12/15LO gene protects mice from lesion formation, while transgenic mice that over-express 12/15LO are more prone to lesion development. We hypothesize that 12/15LO activity in the macrophage may have a “pro-atherogenic” effect by impairing the ABCG1 cholesterol secretory pathway. We recently found that J774 macrophages that stably over-express 12/15LO have a 20% reduction in cholesterol efflux to HDL compared to mock transfected controls. Likewise, treatment of J774 macrophages with the 12/15LO product 12SHETE (500nm) produced a similar 20% reduction in cholesterol efflux to HDL. Concurrently with the reduction in cholesterol efflux, we found a significant 2.5-fold reduction in ABCG1 protein expression following 4 hour treatment with 12SHETE (500nM). 12/15LO over-expressing J774 macrophages also had a significant 2-fold reduction in ABCG1 protein expression. We found that there was no change in mRNA expression in 12/15LO over-expressing J774 macrophages or macrophages treated with 12SHETE (500nM), and thus mRNA changes could not account for the reduction in protein. 12SHETE, however, did increase ABCG1 protein turn-over when blocked by cycloheximide. Thus, we conclude that 12/15LO activity reduces ABCG1 expression and function in macrophages by enhancing the degradation rate of ABCG1. We hypothesize that changes in ABCG1 phosphorylation may lead to enhanced 12/15LO-mediated protein degradation. J774 macrophages were treated with 12SHETE (500nM) for 4 hours in the presence of PKC isoform inhibitors (10μM). PKC zeta pseudosubstrate, but not Bisindolylmaleimide, Gö6976, or Rottlerin, restored ABCG1 levels indicating that PKC zeta may phosphorylate and target ABCG1 for enhanced degradation. An understanding of the mechanisms by which 12/15LO increases ABCG1 degradation will aid in developing beneficial therapies that target this cholesterol removal pathway.