Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids
MEK1/2 inhibitors activate macrophage ABCG1 expression and reverse cholesterol transport—An anti-atherogenic function of ERK1/2 inhibition
Introduction
Atherosclerosis is one of major causes for coronary heart diseases (CHD), and a chronic pathological process with disorders in lipid metabolism and inflammation [1]. Recruitment of monocytes, subsequent monocyte/macrophage differentiation in arteries and formation of lipid-laden macrophage/foam cells are critical steps in the development of lesions [2]. Macrophage cholesterol homeostasis depends on cholesterol uptake and free cholesterol efflux which are mediated by multiple molecules [3], [4]. The un-controlled cholesterol uptake by scavenger receptors, such as CD36, facilitates foam cell formation. In contrast, foam cell formation is inhibited by cholesterol efflux from macrophages. Macrophage cholesterol efflux and reverse cholesterol transport (RCT) can substantially reduce atherosclerosis. The ATP-binding cassette transporter G1 (ABCG1), a member of the ABC transporter family, mediates cholesterol efflux from macrophage/foam cells to the extracellular cholesterol acceptor, high-density lipoprotein (HDL) [5], [6]. Deficiency of ABCG1 expression can disrupt lipid homeostasis and enhance lesion development in LDL receptor deficient (LDLR−/−) mice [7]. The combined deficiency of ABCG1 and ATP-binding cassette transporter A1 (ABCA1) expression can promote foam cell formation and atherosclerosis in both wild type and LDLR−/− mice [8], [9]. However, some variable effects of ABCG1 on cholesterol efflux or atherosclerosis have also been reported. For example, in lipid-loaded THP-1 macrophages, inhibition of ABCG1 expression by siRNA has little effect on LXR ligand-activated cholesterol efflux [10]. Deficiency of ABCG1 expression increases lesions at the early stage while decreasing lesion at the advanced stage of atherosclerosis in LDLR−/− mice [11].
Similar to ABCA1, ABCG1 expression is transcriptionally activated by the liver X receptor (LXR), a ligand-activated transcription factor [12]. Several oxysterols, such as 24S-hydroxycholesterol and 25-hydroxycholesterol, serve as endogenous LXR ligands [13]. Meanwhile, some synthetic ligands, such as T0901317 and GW3965, have been demonstrated to potentiate LXR activity [14]. LXR activation by its ligand can lead to formation of a heterodimer of LXR with another nuclear protein, retinoid X receptor (RXR). The complex of LXR/RXR binds to the LXR responsive element (LXRE) in the promoter of LXR target genes thereby activating their transcription [12]. The LXRE is also called DR4 because the six conserved nucleotides are repeated in this motif (AGGTCAN4AGGTCA) separated by any four nucleotides.
Extracellular signal-regulated kinases 1/2 (ERK1/2) can be activated by different growth stimuli through a kinase cascade in which both Thr/Tyr residues in ERK1/2 are phosphorylated by MAP kinase kinases 1/2 (MEK1/2) [15]. ERK1/2 activity is involved in a variety of cellular activities, such as differentiation, proliferation, immune response and metabolism [16]. In the vascular system, ERK1/2 has been determined to be highly expressed and activated in lesion areas of cholesterol-fed rabbits [17]. We previously reported that inhibition of ERK1/2 by MEK1/2 inhibitors synergized LXR ligand-induced ABCA1 expression and cholesterol efflux to apoAI, another extracellular cholesterol acceptor [18]. Furthermore, we showed that the combination of MEK1/2 inhibitor (U0126) and LXR ligand (T0901317) can synergistically reduce atherosclerosis in apoE deficient (apoE−/−) mice without the adverse effects of hepatic lipogenesis and hypertriglyceridemia which is induced by T0901317 alone [19]. These findings indicate that ERK1/2 activity is involved in the development of atherosclerosis, while MEK1/2 inhibitors may have multiple anti-atherogenic functions. Therefore, in this study we investigated the effect of MEK1/2 inhibitors on macrophage ABCG1 expression and functions.
Section snippets
Materials
PD98059 and U0126 were purchased from LC laboratories (Woburn, MA). Resveratrol, nicotinamide and 9-cis-retinoic acid (9-cis-RA) were purchased from Sigma-Aldrich (St Louis, MO). Both low-density lipoprotein (LDL) and HDL were purchased from Athens Research & Technology, Inc. (Athens, GA). Acetylated LDL (AcLDL) was prepared as described [20]. Anti-ABCG1 (Cat #: 13578-1-AP) and LXR rabbit polyclonal antibodies were purchased from Proteintech Group (Chicago, IL). Anti-SIRT1 rabbit polyclonal and
MEK1/2 inhibitors activate macrophage ABCG1 protein expression
We initially treated RAW264.7 macrophages with MEK1/2 inhibitors, PD98059 and U0126, overnight and determined ABCG1 protein expression by Western blot. Fig. 1A demonstrates that both PD98059 and U0126 induced ABCG1 expression at different concentrations with the maximum induction at 10 μM (PD98059) and 0.5 μM (U0126), respectively. The time course study (Fig. 1B) indicates that induction of ABCG1 expression can last for 24 h after treatment with a faster induction by U0126 than PD98059.
Treatment
Discussion
In the animal models, overexpression and activation of ERK1/2 in atherosclerotic lesions of cholesterol-fed rabbits has been determined, while U0126 synergizes T0901317-inhibited atherosclerosis with elimination of LXR-induced deleterious effects in apoE−/− mice [17], [19]. These studies directly demonstrate the involvement of ERK1/2 in atherosclerosis, and the anti-atherogenic properties of MEK1/2 inhibitors. However, the underlying mechanisms for the anti-atherogenic properties of ERK1/2
Conflict of interest
None declared.
Transparency document
Acknowledgements
This work was supported by the National Science Foundation of China (NSFC) Grants 81473204 to JH, 81573427 to YD, 31400694 to YC and 81503064 to LZ; the Grant of Tianjin Municipal Science and Technology Commission of China (No. 16JCZDJC134700), the Program for Changjiang Scholars and Innovative Research Team in University (No. IRT13023), and the 111 Project (B08011) to JH; the International Science & Technology Cooperation Program of China2015DFA30430 to JH, YD and YC.
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These authors contributed equally to this work.