MEK1/2 inhibitors activate macrophage ABCG1 expression and reverse cholesterol transport—An anti-atherogenic function of ERK1/2 inhibition

https://doi.org/10.1016/j.bbalip.2016.06.017Get rights and content

Highlights

  • MEK1/2 inhibitors activate ABCG1 expression and cholesterol efflux in vitro.

  • MEK1/2 inhibitors enhance RCT and inhibit aortic sinus lesions in vivo.

  • MEK1/2 inhibitors activate ABCG1 expression by regulating multiple pathways.

  • Increased ABCG1 expression contributes to reduction of lesions by MEK1/2 inhibitors.

Abstract

Expression of ATP-binding cassette transporter G1 (ABCG1), a molecule facilitating cholesterol efflux to HDL, is activated by liver X receptor (LXR). In this study, we investigated if inhibition of ERK1/2 can activate macrophage ABCG1 expression and functions. MEK1/2 inhibitors, PD98059 and U0126, increased ABCG1 mRNA and protein expression, and activated the natural ABCG1 promoter but not the promoter with the LXR responsive element (LXRE) deletion. Inhibition of ABCG1 expression by ABCG1 siRNA did enhance the formation of macrophage/foam cells and it attenuated the inhibitory effect of MEK1/2 inhibitors on foam cell formation. MEK1/2 inhibitors activated macrophage cholesterol efflux to HDL in vitro, and they enhanced reverse cholesterol transport (RCT) in vivo. ApoE deficient (apoE−/−) mice receiving U0126 treatment had reduced sinus lesions in the aortic root which was associated with activated macrophage ABCG1 expression in the lesion areas. MEK1/2 inhibitors coordinated the RXR agonist, but not the LXR agonist, to induce ABCG1 expression. Furthermore, induction of ABCG1 expression by MEK1/2 inhibitors was associated with activation of SIRT1, a positive regulator of LXR activity, and inactivation of SULT2B1 and RIP140, two negative regulators of LXR activity. Taken together, our study suggests that MEK1/2 inhibitors activate macrophage ABCG1 expression/RCT, and inhibit foam cell formation and lesion development by multiple mechanisms, supporting the concept that ERK1/2 inhibition is anti-atherogenic.

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.

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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.

References (43)

  • P. Wood et al.

    Ras/mitogen-activated protein kinase (MAPK) signaling modulates protein stability and cell surface expression of scavenger receptor SR-BI

    J. Biol. Chem.

    (2011)
  • D.A. Mogilenko et al.

    PPARgamma activates ABAC1 gene transcription but reduces the level of ABCA1 protein in HepG2 cells

    Biochem. Biophys. Res. Commun.

    (2010)
  • D.N. Patel et al.

    Interleukin-17 stimulates C-reactive protein expression in hepatocytes and smooth muscle cells via p38 MAPK and ERK1/2-dependent NF-kappaB and c/EBPbeta activation

    J. Biol. Chem.

    (2007)
  • Y. Li et al.

    SIRT1 inhibition reduces IGF-I/IRS-2/RAS/ERK1/2 signaling and protects neurons

    Cell Metab.

    (2008)
  • N. Terasaka et al.

    T-0901317, a synthetic liver X receptor ligand, inhibits development of atherosclerosis in LDL receptor-deficient mice

    FEBS Lett.

    (2003)
  • V. Fuster et al.

    Low priority of cardiovascular and chronic diseases on the global health agenda: a cause for concern

    Circulation

    (2007)
  • R. Ross

    The pathogenesis of atherosclerosis: a perspective for the 1990s

    Nature

    (1993)
  • W.J. de Villiers et al.

    Macrophage scavenger receptors and foam cell formation

    J. Leukoc. Biol.

    (1999)
  • K. Simons et al.

    How cells handle cholesterol

    Science

    (2000)
  • N. Wang et al.

    ATP-binding cassette transporters G1 and G4 mediate cellular cholesterol efflux to high-density lipoproteins

    Proc. Natl. Acad. Sci. U. S. A.

    (2004)
  • N. Wang et al.

    LXR-induced redistribution of ABCG1 to plasma membrane in macrophages enhances cholesterol mass efflux to HDL

    Arterioscler. Thromb. Vasc. Biol.

    (2006)
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    These authors contributed equally to this work.

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