The lipid moiety 7-ketocholesteryl-9-carboxynonanoate mediates binding interaction of oxLDL to LOX-1 and upregulates ABCA1 expression through PPARγ
Introduction
Atherosclerosis, a major cause of cardiovascular disease, can be considered both a lipid metabolic disorder and a chronic inflammatory disease [1], [2]. Macrophage-derived foam cell formation is a hallmark of the initial stages of atherosclerosis. Uncontrolled uptake of oxidized low-density lipoprotein (oxLDL), excessive cholesterol esterification and/or reduced cholesterol efflux result in cholesterol ester accumulation in the cytoplasm of macrophages and the generation of foam cells [3], which contribute to atherosclerotic plaque formation and progression [4], [5].
In macrophages, several scavenger receptors (SRs), including CD36, SR-A1 and Lectin-like oxLDL receptor-1 (LOX-1), mediate oxLDL uptake and foam cell formation [3], [4]. LOX-1, a specific membrane receptor for oxLDL, can recognize and bind to oxLDL on the surface of macrophages [6], [7]. LOX-1 consists of four domains: a short N-terminal cytoplasmic domain, a transmembrane domain, a neck region that controls receptor oligomerization and an extracellular C-type lectin-like domain (CTLD) [6], [7]. The crystal structure has been elucidated and shows that the basic spine located at the CTLD binds the negatively charged lipid molecules and is responsible for oxLDL recognition [8], [9], [10]. Recent studies have demonstrated that LOX-1 is involved in the uptake of oxLDL by macrophages. In normal conditions, the effect of LOX-1 on intake and catabolism of oxLDL by macrophages is small. This is likely due to the high expression of other SRs, such as SR-AI/II, SR-BI and cluster of differentiation 36 (CD36), which could mask the contribution of LOX-1. However, when macrophages are stimulated by pro-inflammatory cytokines or oxLDL, LOX-1 is significantly upregulated, and the internalization of oxLDL increases by > 40%, indicating that LOX-1 plays an important role in the endocytic uptake and accumulation of oxLDL [4], [11], [12], [13].
Peroxisome proliferator-activated receptor gamma (PPARγ), a nuclear receptor, has a critical function in maintaining cholesterol homeostasis [14]. The activation and upregulation of PPARγ can increase the expression of liver X receptor α (LXRα) and ATP-binding cassette transporter A1 (ABCA1), leading to cellular cholesterol efflux [15], [16], [17]. Furthermore, LOX-1 was reported to specifically activate the PPARγ signalling pathway following oxLDL stimulation, suggesting that LOX-1 is closely associated with PPARγ signal transduction and involved in cholesterol efflux [18].
In our previous study, we purified a negatively charged lipid moiety from oxLDL, 7-ketocholesteryl-9-carboxynonanoate (oxLig-1) [19], [20], and found that the oxLig-1 upregulated LXRα and ABCA1 via the PPARγ signalling pathway [21]. However, the underlying mechanisms, whereby ligand binding to the receptor triggers activation of PPARγ signalling events, have remained unclear. As the CTLD of LOX-1 could bind negatively charged lipid components [8], [9], [10], we hypothesized that oxLig-1 binds LOX-1. Additionally, LOX-1 can specifically activate the PPARγ signalling pathway following oxLDL stimulation [18]. Therefore, we further speculated that the binding of oxLig-1 to LOX-1 promotes upregulation of ABCA1 expression through PPARγ signal transduction. To assess our hypothesis, we investigated the mechanism underlying oxLig-1 binding to LOX-1 and determined the effect of this binding interaction on upregulation of ABCA1 mediated by PPARγ.
Section snippets
Chemicals and reagents
Azelaic acid, azelaic acid monomethyl ester, cholesterol, cholest-5-en-7-one, 5-cholesten-3β-ol-7-one (7-ketocholesterol), 4-dimethylaminopyridine (DMAP) and N,N′-dicyclohexylcarbodiimide (DCC) were obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Antibodies were obtained from the following sources. Anti-LOX-1 (ab60178) and anti-LXRα (ab176323) antibodies were purchased from Abcam (USA). Anti-PPARγ (sc-7196) and anti-GAPDH (sc-25778) antibodies were obtained from Santa Cruz (USA).
Molecular docking of oxLig-1 with LOX-1
The LOX-1 CTLD has been reported to be responsible for the binding of oxLDL [8], [9], [10]. To investigate the binding interaction between oxLig-1 and LOX-1, we docked oxLig-1, me-oxLig-1, and cholest-5-en-7-one with the CTLD of LOX-1. As shown in Fig. 1A, the binding groove formed by the hydrophobic amino acids established an excellent hydrophobic environment for the insertion of oxLig-1, and the positively charged residues Arg 208, Arg 229, and Arg 231 were located on either side of the
Discussion
LOX-1 is intimately involved in accumulation of oxLDL and formation of foam cells. In the initial stage, the critical step is recognition and binding of oxLDL by LOX-1 receptors. Recent studies have shown that the binding interaction between oxLDL and LOX-1 is predominantly mediated by oxidized phospholipids [8], [29]. In this study, we first determined that oxLig-1, an oxidation product of cholesteryl linoleate derived from oxLDL, can act as a novel ligand to mediate the binding of oxLDL to
Conflict of interest
The authors declare no conflicts of interest.
Acknowledgements
This work was supported by grants from the National Natural Science Foundation of China (No: 81270361, 30971232 and 81202536).
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These authors contributed equally to this work.