Inhibition of Mitogen-Activated Protein Kinase Erk1/2 Promotes Protein Degradation of ATP Binding Cassette Transporters A1 and G1 in CHO and HuH7 Cells

Vishwaroop Mulay,Peta Wood,Anna Álvarez-Guaita,Meritxell Reverter,Carlos Enrich,Carles Rentero,Melanie Manetsch,Kerry-Anne Rye,Thomas Grewal,Karen Cecilia Chan,Masoud Darabi,Rose Cairns,Monira Hoque,Joerg Heeren

doi:10.1371/journal.pone.0062667

Abstract

Signal transduction modulates expression and activity of cholesterol transporters. We recently demonstrated that the Ras/mitogen-activated protein kinase (MAPK) signaling cascade regulates protein stability of Scavenger Receptor BI (SR-BI) through Proliferator Activator Receptor (PPARα) -dependent degradation pathways. In addition, MAPK (Mek/Erk 1/2) inhibition has been shown to influence liver X receptor (LXR) -inducible ATP Binding Cassette (ABC) transporter ABCA1 expression in macrophages. Here we investigated if Ras/MAPK signaling could alter expression and activity of ABCA1 and ABCG1 in steroidogenic and hepatic cell lines. We demonstrate that in Chinese Hamster Ovary (CHO) cells and human hepatic HuH7 cells, extracellular signal-regulated kinase 1/2 (Erk1/2) inhibition reduces PPARα-inducible ABCA1 protein levels, while ectopic expression of constitutively active H-Ras, K-Ras and MAPK/Erk kinase 1 (Mek1) increases ABCA1 protein expression, respectively. Furthermore, Mek1/2 inhibitors reduce ABCG1 protein levels in ABCG1 overexpressing CHO cells (CHO-ABCG1) and human embryonic kidney 293 (HEK293) cells treated with LXR agonist. This correlates with Mek1/2 inhibition reducing ABCG1 cell surface expression and decreasing cholesterol efflux onto High Density Lipoproteins (HDL). Real Time reverse transcriptase polymerase chain reaction (RT-PCR) and protein turnover studies reveal that Mek1/2 inhibitors do not target transcriptional regulation of ABCA1 and ABCG1, but promote ABCA1 and ABCG1 protein degradation in HuH7 and CHO cells, respectively. In line with published data from mouse macrophages, blocking Mek1/2 activity upregulates ABCA1 and ABCG1 protein levels in human THP1 macrophages, indicating opposite roles for the Ras/MAPK pathway in the regulation of ABC transporter activity in macrophages compared to steroidogenic and hepatic cell types. In summary, this study suggests that Ras/MAPK signaling modulates PPARα- and LXR-dependent protein degradation pathways in a cell-specific manner to regulate the expression levels of ABCA1 and ABCG1 transporters.

Highlights

Anti-atherosclerotic properties of High Density Lipoproteins (HDL) and apolipoprotein A-I include their ability to promote reverse cholesterol transport (RCT), the removal of excess cholesterol from peripheral tissues to the liver for bile secretion [1,2,3]
We showed that MAPK/Erk kinase 1 (Mek1)/2 inhibition reduced PPARa-dependent Scavenger Receptor BI (SR-BI) protein stability in Chinese Hamster Ovary (CHO), human embryonic kidney 293 (HEK293) and hepatic HuH7 cells, but not peripheral cells, such as bovine aortic endothelial (BAEC) and monocytic (THP1) cell lines [28]
To determine if the Ras/mitogen-activated protein kinase (MAPK) pathway could alter ABCA1 protein levels, possibly in a PPARa-dependent manner, we first examined CHOwt cells, which have been proven a valuable model to study the involvement of ABCA1 in cholesterol transport [36,37], exhibit HDL-inducible Ras/MAPK activity, Ras/MAPK-inducible PPARa phosphorylation and SRBI expression [25,26,27,28]

Summary

Introduction

Anti-atherosclerotic properties of HDL and apolipoprotein A-I (apoA-I) include their ability to promote reverse cholesterol transport (RCT), the removal of excess cholesterol from peripheral tissues to the liver for bile secretion [1,2,3]. An increasing number of studies suggest that cell surface binding and internalization of HDL and apo-AI activate signaling proteins such as protein kinase A and C (PKA, PKC), Rac/Rho GTPases, Janus Kinase 2 (JAK2), calmodulin and MAPK to modulate the ability of cells to export cholesterol [4,5,6]. Given their potential as pharmaceutical targets, the control of ABC transporter and SR-BI expression received great attention, and transcriptional upregulation of ABCA1, ABCG1 and SR-BI via nuclear receptors, including LXR, PPARa and PPARc, is well established [7,8]. Little is known about ABCG1 protein turnover, but ubiquitination as well as calpain have recently been identified as influencing ABCG1 protein levels in macrophages [11,19,20,21]

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