Overexpression of mitochondrial cholesterol delivery protein, StAR, decreases intracellular lipids and inflammatory factors secretion in macrophages
Introduction
Macrophages play a key role in both the initiation and progression of atherosclerosis. Macrophages in arterial walls uptake lipoproteins, especially oxidized low-density lipoprotein (ox-LDL), and accumulate cholesterol and other neutral lipids resulting in foam cell formation, which is considered a critical process in the development of atherosclerosis [1], [2]. The factors regulating net accumulation or elimination of lipids from macrophages are critical in formation of the early atherosclerotic lesion and progression to the chronic stage of atherosclerosis. To stimulate the pathways involved in decreasing lipids in macrophages may be an effective way to prevent atherosclerosis.
Several studies have shown that the processes of uptake and/or efflux of cholesterol from macrophages are regulated by nuclear receptors, including liver X receptors (LXRs) and proliferation peroxysome activator receptors (PPARs). Expression of a number of ATP-binding cassette transporters (ABCs), such as ABCA1, ABCG1, and ABCG4 involved in lipid efflux are regulated through activation of LXRs [3]. ABC transporters have been implicated in macrophage reverse cholesterol transport (RCT) and atherosclerosis [4], while PPARs have been reported to coordinate the effect of LXRs [5], [6] and play an important role in inflammatory response through interference with pro-inflammatory transcription factor pathways [7].
Oxysterols are well known as physiological ligands for LXRs [8]. 22-Hydroxycholesterol and 27-hydroxycholesterol synthesized by mitochondrial sterol 27-hydroxylase (CYP27A1) are able to activate LXRs as endogenous ligands in cholesterol-loaded cells [9], [8]. CYP27A1 activity has been reported to play a fundamental role in the maintenance of intracellular lipid homeostasis in vitro and in vivo [10], [11]. Recent studies have shown that mitochondrial cholesterol delivery by steroidogenic acute regulatory (StAR) protein is the rate-limiting step for CYP27A1 activity [12], [13], [14]. Overexpression of StAR can increase the activity of CYP27A1 to produce regulatory oxysterols that appear capable of regulating intracellular lipid homeostasis [13]. More recent studies have shown that overexpression of StAR in primary human and rat hepatocytes dramatically increases oxysterols in hepatocyte nuclei. These oxysterols are potent regulators involved in intracellular lipid metabolism [15], [16], [17]. Our previous work showed that StAR mRNA and protein also expressed in macrophages [18]. However, the physiological significance of StAR in maintenance of cholesterol homeostasis in macrophages remains unknown.
In the present study, we show that overexpression of StAR decreases intracellular lipid levels and the secretion of inflammatory factors. These results suggest that the mitochondrial cholesterol delivery protein StAR plays an important role in lipid metabolism and the inflammatory response in human THP-1 derived macrophages.
Section snippets
Materials and methods
All cell culture media and additives, TRIZOL reagent and SuperScript TMIII First-Strand Synthesis System for RT-PCR were purchased from Invitrogen (Carlsbad, CA). SYBR® Green real-time PCR Master Mix was from Toyobo Company (Osaka, JP). The primary antibodies against StAR and β-actin were purchased from Abcam (Cambridge, MA). ABCA1 and Histone H4 antibodies were from United States Biological (Swampscott, MA) and Cell Signaling Technology, Inc. (Boston, MA), respectively. ABCG1, CYP27A1, PPARγ
Recombinant StAR gene was successfully overexpressed by transfection of recombinant adenovirus
Infection of THP-1 derived macrophages with recombinant adenovirus containing a CMV-driven gene encoding StAR produced high StAR mRNA and protein levels without inducing any evidence of cell toxicity. Real-time RT-PCR analysis showed that infection with Adv-CMV-StAR increased StAR mRNA levels by 130-fold in the cells (Supplemental Fig. 1A). Western blot analysis of mitochondrial proteins showed one major immunoreactive band with a molecular weight of 30 kDa (lower bands) and a second band with
Discussion
Hyperlipidemia is one of the most important risk factors for atherosclerosis, in which macrophages are important cells that can uptake extra cholesterol and enhance the local inflammatory response, which promotes the progression of atherosclerosis [26]. In the current study, we overexpressed StAR in THP-1 derived macrophages by recombinant adenovirus infection. After successful infection, intracellular neutral lipid levels in the macrophages were significantly decreased and total cholesterol
Acknowledgements
This work was supported by Grant (30470689) from the National Foundation for Natural Science, PR China for Yin, L., and by National Institute of Health, USA, Grant R01 HL078898 and VA Merit Review Grant for Ren, S.
References (31)
Monocyte recruitment and foam cell formation in atherosclerosis
Micron
(2006)- et al.
Characterization of the human ABCG1 gene: liver X receptor activates an internal promoter that produces a novel transcript encoding an alternative form of the protein
J Biol Chem
(2001) - et al.
A PPAR gamma-LXR-ABCA1 pathway in macrophages is involved in cholesterol efflux and atherogenesis
Mol Cell
(2001) - et al.
27-Hydroxycholesterol is an endogenous ligand for liver X receptor in cholesterol-loaded cells
J Biol Chem
(2001) - et al.
Role of CYP27A in cholesterol and bile acid metabolism
J Lipid Res
(2005) - et al.
Detection of the steroidogenic acute regulatory protein, StAR, in human liver cells
Biochim Biophys Acta
(2005) - et al.
Transport of cholesterol into mitochondria is rate-limiting for bile acid synthesis via the alternative pathway in primary rat hepatocytes
J Biol Chem
(2002) - et al.
Biosynthesis of the regulatory oxysterol, 5-cholesten-3beta,25-diol 3-sulfate, in hepatocytes
J Lipid Res
(2007) - et al.
Identification of a novel sulfonated oxysterol, 5-cholesten-3beta,25-diol 3-sulfonate, in hepatocyte nuclei and mitochondria
J Lipid Res
(2006) - et al.
Sulfated oxysterol, 25HC3S, is a potent regulator of lipid metabolism in human hepatocytes
Biochem Biophys Res Commun
(2007)