Abstract 230: Stimulation of Cholesterol Efflux from Human Macrophage by Liver X Receptor Agonists is a 2-Step Mechanism Requiring ARL7 and ABCA1

Nuclear Liver X Receptors activation by synthetic agonists was proven to be atheroprotective in mice; an effect likely based on stimulation of cellular cholesterol efflux from arterial macrophages. However, mechanisms involved in free cholesterol efflux from mouse macrophages appear distinct from those operating in human macrophages. The objective of this study was to decipher precise cellular mechanisms controlling free cholesterol efflux from human macrophages upon LXR stimulation. In THP-1 and human monocyte-derived macrophages (HMDM), treatment with the LXR agonist GW3965 efficiently induced ARL7 expression (6-fold, p<0.05), an effect associated with an increased amount of plasma membrane free cholesterol available for efflux (+25%, p<0.05) and a higher lipid rafts formation (+10%, p<0.05). Both effects were abolished in ARL7 Knockdown (KD) macrophages, leading to a lack of stimulation of cholesterol efflux by GW3965. Specific targeting of each LXR isoforms, LXRα and LXRβ, by RNAi revealed that LXRα silencing in THP-1 and HMDM reduced significantly expression of cholesterol transporters ABCA1, ABCG1 and receptor SR-BI/Cla-1 mRNA levels, as well as free cholesterol efflux to apoA1 (-30%, p<0.05) and to HDL (-20%, p<0.05) upon stimulation with LXR, whereas LXRβ silencing has no impact. Interestingly, stimulation of cholesterol efflux to HDL by GW3965 was significantly reduced (-50%, p<0.05) in ABCA1 KD THP-1 macrophages; those cells being incapable to promote cholesterol efflux to apoA1. However, silencing of ABCG1 or SR-B1/Cla-1 had no impact on cholesterol efflux to HDL from either control or ABCA1 KD THP-1 macrophages treated or not with LXR agonist. By contrast stimulation of cholesterol efflux to HDL by GW3965 was completely abolished in LXRα/ABCA1 double KD macrophages, highlighting the major contribution of ABCA1 in cholesterol efflux from human macrophage. We conclude that LXR-mediated stimulation of cholesterol efflux from human macrophages is a two-steps mechanism. First, LXR activation promotes ARL7-dependent free cholesterol transport to plasma membrane, mostly in lipid raft domains. Then, membrane free cholesterol is exported to apoA1 and HDL acceptors through ABCA1; this latter step being controlled selectively by LXRα.