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J. Biol. Chem., Vol. 280, Issue 23, 22212-22221, June 10, 2005

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Regulation of Macrophage Cholesterol Efflux through Hydroxymethylglutaryl-CoA Reductase Inhibition

A ROLE FOR RhoA IN ABCA1-MEDIATED CHOLESTEROL EFFLUX^*

Carmen A. Argmann, Jane Y. Edwards¶, Cynthia G. Sawyez¶, Caroline H. O'Neil, Robert A. Hegele¶||**, J. Geoffrey Pickering¶||, and Murray W. Huff¶||

From the Robarts Research Institute Vascular Biology Group and the Departments of ^¶Medicine and Biochemistry, the University of Western Ontario, London, Ontario N6A 5K8, Canada

Received for publication, March 14, 2005

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The cholesterol biosynthetic pathway produces numerous signaling molecules. Oxysterols through liver X receptor (LXR) activation regulate cholesterol efflux, whereas the non-sterol mevalonate metabolite, geranylgeranyl pyrophosphate (GGPP), was recently demonstrated to inhibit ABCA1 expression directly, through antagonism of LXR and indirectly through enhanced RhoA geranylgeranylation. We used HMG-CoA reductase inhibitors (statins) to test the hypothesis that reduced synthesis of mevalonate metabolites would enhance cholesterol efflux and attenuate foam cell formation. Preincubation of THP-1 macrophages with atorvastatin, dose dependently (1–10 µM) stimulated cholesterol efflux to apolipoprotein AI (apoAI, 10–60%, p < 0.05) and high density lipoprotein (HDL₃) (2–50%, p < 0.05), despite a significant decrease in cholesterol synthesis (2–90%). Atorvastatin also increased ABCA1 and ABCG1 mRNA abundance (30 and 35%, p < 0.05). Addition of mevalonate, GGPP or farnesyl pyrophosphate completely blocked the statin-induced increase in ABCA1 expression and apoAI-mediated cholesterol efflux. A role for RhoA was established, because two inhibitors of Rho protein activity, a geranylgeranyl transferase inhibitor and C3 exoenzyme, increased cholesterol efflux to apoAI (20–35%, p < 0.05), and macrophage expression of dominant-negative RhoA enhanced cholesterol efflux to apoAI (20%, p < 0.05). In addition, atorvastatin increased the RhoA levels in the cytosol fraction and decreased the membrane localization of RhoA. Atorvastatin treatment activated peroxisome proliferator activated receptor and increased LXR-mediated gene expression suggesting that atorvastatin induces cholesterol efflux through a molecular cascade involving inhibition of RhoA signaling, leading to increased peroxisome proliferator activated receptor activity, enhanced LXR activation, increased ABCA1 expression, and cholesterol efflux. Finally, statin treatment inhibited cholesteryl ester accumulation in macrophages challenged with atherogenic hypertriglyceridemic very low density lipoproteins indicating that statins can regulate foam cell formation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Arterial macrophages are faced with the task of internalizing and metabolizing atherogenic lipoproteins, an event that challenges cellular cholesterol homeostasis (1–3). Cholesterol homeostasis is maintained through the coordinated regulation of pathways mediating cholesterol uptake, storage, de novo synthesis, and efflux, and it is likely the dysregulation of these signals that promotes foam cell formation (2–4). In response to lipoprotein uptake, regulation of cellular cholesterol metabolism is accomplished, in part, by the nuclear hormone receptors peroxisome proliferator activated receptor (PPAR)¹, PPAR, and the liver X receptor (LXR), ligand-activated transcription factors that target genes involved in lipid and lipoprotein metabolism (reviewed in Refs. 5 and 6). A major source of endogenous ligands for these receptors are fatty acid derivatives and oxysterols derived from the cholesterol biosynthetic pathway (7) or lipoprotein cholesterol (8, 9). This pathway also yields non-sterol mevalonate metabolites involved in isoprenoid synthesis that have until recently mainly been associated with the regulation of cell cycle control and cytoskeleton organization (9). However, isoprenoid intermediates have been shown to affect PPAR, PPAR, and LXR activation (10–14), suggesting they may have a role in the regulation of lipid homeostasis and potentially macrophage foam cell formation.

The committed step in the biosynthesis of cholesterol and isoprenoids is catalyzed by 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, which promotes the deacylation of HMG-CoA to mevalonate (see Fig. 1) (9, 15). This pathway produces numerous bioactive signaling molecules including oxysterols, farnesyl pyrophosphate (FPP), and geranylgeranyl pyrophosphate (GGPP), which regulate transcriptional and posttranscriptional events that affect various biological processes (9). Oxysterols regulate the concentration of cellular sterols by activating LXR (16) and by controlling the activities of HMG-CoA reductase and the sterol-sensing transcription factors, sterol regulatory element-binding protein (SREBP)-1 and SREBP-2. Oxysterols are hypothesized to regulate the latter two activities by causing influx of plasma membrane cholesterol into the endoplasmic reticulum membrane. In turn this facilitates the interaction of HMG-CoA reductase or the SREBP cleavage activating protein (SCAP) with the Insig proteins (17, 18). Interaction with Insig is required for the cholesterol-regulated degradation of HMG-CoA reductase, whereas interaction of Insig with SCAP retains the SCAP·SREBP complex within the endoplasmic reticulum, preventing activation of SREBPs to their active processed forms.

View larger version (45K): [in this window] [in a new window]   FIG. 1. The regulation of macrophage cholesterol efflux by HMG-CoA reductase inhibition. Statins inhibit the rate-limiting step in cholesterol biosynthesis reducing the production of mevalonate and mevalonate-intermediates. Statins also reduce isoprenoid synthesis, which results in the inhibition of the geranylgeranylation of small GT-Pases, including RhoA. Consequently, the activation of Rho-effector proteins, such as the Rho-associated kinases, are inhibited. One consequence is increased PPAR activity; ABC expression is ultimately increased through LXR activation. The statin-mediated increase in cholesterol efflux was reversed by mevalonate and the isoprenoid intermediates, FPP and GGPP. Inhibitors of Rho protein activity, GGTI and C3 exoenzyme, mimicked the effect of statins on cholesterol efflux. The PPAR antagonist (GW9662) and LXR antagonist (ECHS) demonstrated that increased PPAR activity leading to LXR activation are involved in the statin-mediated effect. This figure was adapted from Takemoto et al. (29).

Key targets of PPAR and LXR activation are the ATP-binding cassette (ABC) proteins, ABCA1 and ABCG1 (3, 23, 24). ABCA1 controls the rate-limiting step in cellular cholesterol and phospholipid efflux to apoA1 (25), and ABCG1 facilitates cholesterol efflux from macrophages to HDL (26). Net cholesteryl ester (CE) deposition in macrophages depends on the competing processes of lipoprotein uptake and cholesterol efflux. The integral role of ABCA1 in foam cell formation is exemplified by the loss-of-function mutations causing Tangier disease, characterized by a significant accumulation of cholesterol in macrophages (reviewed in Ref. 27). ABCA1 and ABCG1 expression is up-regulated by agonists of PPAR and LXR through a transcriptional cascade ultimately dependent on the activation of LXR (23, 24). We recently reported that PPAR agonists blocked macrophage foam cell formation induced by oxidized lipoproteins through enhanced ABCA1-mediated cholesterol efflux in an LXR-dependent manner (28).

Statins are competitive inhibitors of HMG-CoA reductase that decrease cholesterol biosynthesis and are widely used for cholesterol-lowering therapy and prevention of atherosclerosis-related events (15). A number of pleiotropic effects or properties of statins independent of low density lipoprotein (LDL)-cholesterol-lowering, have been identified in vascular cells which include: improved endothelial function; enhanced plaque stability; decreased oxidative stress and inflammation; and inhibition of thrombogenic responses in the vascular wall (reviewed in Ref. 29). Some of these effects have been associated with the ability of statins to decrease isoprenoid synthesis and subsequent prenylation of small GTP-binding proteins such as Rho and Ras (29, 30).

In the present study we used HMG-CoA reductase inhibitors to test the hypothesis that non-sterol derivatives arising from mevalonate biotransformation play a role in macrophage cholesterol efflux. Treatment of macrophages with the HMG-CoA reductase inhibitor, atorvastatin, significantly enhanced mRNA expression of ABCA1 and ABCG1 and increased cholesterol efflux to apolipoprotein AI (apoAI) and HDL_3. These results occurred despite the statin-induced inhibition of endogenous cholesterol synthesis. The effects of atorvastatin were completely reversible by the addition of exogenous mevalonate and the non-sterol intermediate GGPP. Furthermore, inhibitors of farnesylation and geranylgeranylation each mimicked the effect of statins, supporting the concept that non-sterol mevalonate metabolites, specifically the isoprenoid intermediates, can regulate macrophage cholesterol efflux. The mechanism for these effects was, in part, because of reduced RhoA activation, leading to the activation of LXR. Increased PPAR activity contributed to the enhanced LXR activity. Furthermore, in macrophages challenged with native atherogenic hypertriglyceridemic (HTG) very low density lipoproteins (VLDL), CE accumulation was significantly reduced by incubation with atorvastatin. Therefore, statin-induced inhibition of non-sterol mevalonate intermediates can decrease macrophage foam cell formation through up-regulation of cholesterol efflux.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Lipoproteins—Subjects were recruited from the Outpatient Lipid Clinic at the London Health Sciences Centre, University Campus, London, Ontario. These studies were approved by the University of Western Ontario Health Sciences Standing Committee on Human Research. Large very low density lipoprotein (Sf20–400) were isolated from plasma of type IV hypertriglyceridemic subjects (HTG-VLDL), whereas low density (Sf0–12) and high density lipoproteins (HDL₃) were isolated from normal subjects by sequential ultracentrifugation as previously described (31–33). LDL was acetylated by addition of acetic anhydride (34).

Cell Culture—This study used the human macrophage-like cell line THP-1 (American Type Culture Collection, Rockville, MD), human liver cell line HepG2 (ATCC) (35), and J774A.1 murine macrophages (ATCC) cultured as described previously (32). THP-1 cells were differentiated using phorbol 12,13-dibutyrate at 300 nM for a total of 7 days. For each experiment, the appropriate concentrations of HMG-CoA reductase inhibitors, solubilized in dimethyl sulfoxide (Me₂SO, concentration did not exceed 0.5%), were added to cells. Atorvastatin (sodium salt of the open acid form) was provided by Pfizer and simvastatin (sodium salt of the open acid form) was provided by Merck.

Cholesterol Synthesis—THP-1 macrophages were preincubated for 19 h with increasing doses of atorvastatin (1–10 µM) in RPMI (10% fetal bovine serum) prior to a 5-h incubation of cells in the absence or presence of atorvastatin and 1 µCi of [1-¹⁴C]acetic acid (Amersham Biosciences) in RPMI (5% LPDS (36)). The incorporation of [1-¹⁴C]acetic acid into cholesterol was determined following separation of lipids by thin layer chromatography, as described elsewhere (37).

Cholesterol Efflux Assay—Efflux of cholesterol from cholesterolloaded cells was determined as described previously (38). In brief, THP-1 macrophages were converted to foam cells by incubation with acetylated LDL and [1,2-³H]cholesterol (1 µCi/ml, Amersham Biosciences) for 24 h. The foam cells were subsequently washed, and a statin (atorvastatin or simvastatin) was added to cells in medium containing 0.2% FAF-BSA for 16 h. Cells were then incubated with fresh medium in the presence or absence of statin and containing either 0.2% FAF-BSA only or supplemented with human apoAI (Sigma, 10 µg/ml) or HDL₃ (100 µg protein/ml) for an additional 24 h. The medium was then centrifuged, and the amount of radioactivity was determined. Cholesterol efflux was expressed as the percentage of counts in the medium versus total [³H]cholesterol counts (medium plus cell).

In some experiments, cholesterol efflux was determined in cells that had been preincubated for 16 h with the following compounds prior to a 24 h co-incubation of the compound and acceptor. These included mevalonate (100 µM, Sigma), FPP (5 or 10 µM, Biomol%20Research%20Laboratories">Biomol Research Laboratories Inc.), GGPP (5 or 10 µM, Biomol%20Research%20Laboratories">Biomol Research Laboratories), farnesyl transferase inhibitor (10 µM, Calbiochem, added only in presence of acceptor), geranylgeranyl transferase inhibitor (GGTI, 10 µM, Calbiochem), C3 exoenzyme (10 µg/ml, Calbiochem), the ROCKI/II inhibitor (Y27632, 10 µM, Calbiochem), the PPAR antagonist, bisphenol A diglycidyl ether (39) (10 µM, Sigma), the PPAR antagonist GW9662 (40) (1 µM, Cayman Chemical, Ann Arbor, MI), the LXR agonist TO901317 (41) (1 µM, Sigma), and the LXR antagonist, 5,6-epoxycholesterol-3-sulfate (42) (ECHS, 10 µM, obtained from the Ben May Institute for Cancer Research, University of Chicago, Chicago, Illinois). Mevalonate, FPP, and GGPP were tested both in the absence and presence of atorvastatin, whereas the Rho protein inhibitors were only tested in the absence of a statin. All of the nuclear hormone receptor antagonists used were tested in the absence and presence of a statin, which were added 30 min prior to the addition of the statin.

mRNA Abundance—THP-1 macrophages were seeded at 3.0 x 10⁷ cells/100-mm plate (Falcon Scientific) in RPMI 1640 (10% fetal bovine serum and phorbol dibutyrate, 300 nM). All cells were treated for 24 h in medium supplemented with 5% LPDS in the absence or presence of indicated compound. Total RNA was isolated using TRIzol reagent (Invitrogen) and mRNA abundance was measured as described previously (28). Briefly, oligonucleotides (5 pmol) were 5'-end-labeled with [-³²P]ATP. An oligonucleotide of interest and glyceraldehyde-3-phosphate dehydrogenase oligonucleotide were simultaneously hybridized to total RNA (20 µg) from either control or treated cells and incubated for 16 h. DNA·RNA hybrids were digested with S1 nuclease, precipitated, and separated by denaturing PAGE (19%). Bands were visualized using a PhosphorImager and quantified using Image Quant software (Molecular Dynamics, Sunnyvale, CA).

Analysis of RhoA Cellular Localization—THP-1 macrophages (100-mm dishes) were incubated in the absence or presence of atorvastatin for 24 h (5% LPDS). Cells were scraped into 500 µl of lysis buffer (10 mM Tris/HCl, 10 mM NaCl, 3 mM MgCl₂, 0.5% (octylphenoxy)polyethoxyethanol) containing aprotinin (100 units/ml), ALLN (2 µg/ml), leupeptin (0.1 mM), phenylmethylsulfonyl fluoride (2 mM), pepstatin (5 µg/ml), and benzamidine (250 µg/ml) and homogenized by 15 passes through a 25-gauge needle. The postnuclear supernatant was obtained by centrifugation (500 g, 4 °C, 10 min). The postnuclear fraction was further spun at 100,000 x g for 30 min (4 °C, Beckman TLA 120.2 rotor) to obtain a pellet of the cell membrane fraction and cytosol fraction in the supernatant. The pellet was suspended in membrane resuspension buffer (10 mM Tris/HCl, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% SDS) containing the protease inhibitors. Protein from the cytosol and membrane (20–100 µg) fractions were mixed with SDS loading buffer and subjected to SDS-PAGE on a 12% gel. Proteins were transferred electrophoretically to nitrocellulose membranes, which were blocked (16 h, 4 °C) with 5% (w/v) nonfat dried milk in phosphate-buffered saline. Membranes were incubated with a monoclonal antibody for RhoA (Santa Cruz Biotechnology, Santa Cruz, CA), followed by incubation with a peroxidase-conjugated anti-mouse IgG antibody (Santa Cruz). RhoA was detected using BM chemiluminescence blotting substrate (Roche Diagnostics). Quantification analysis of the developed films was performed using an imaging densitometer (GS-700, Bio-Rad Laboratories, Mississauga, ON, Canada).

Transient Transfection—The coding sequence for human RhoA T19N (Guthrie cDNA Resource Center, Sayre, PA) with the mutation T19N that confers a dominant-negative phenotype, was cloned into a pIRES2-EGFP vector (BD Biosciences Clontech) in the multiple cloning sites with EcoRI and SalI. The fragment encoding RhoT19N-IRES-EGFP was subcloned into the multiple cloning site of pLNCX2 vector (Clontech) using XhoI and NotI restriction sites. The construct was isolated and sequence verified using an ABI 377 prism automated DNA sequencer.

Lipofectamine reagents (Invitrogen) and the manufacturer's protocol were used for transient transfections. THP-1 cells were plated at 4.0 x 10⁶ cells/well in 35-mm wells (Falcon Scientific) in RPMI 1640 (10% fetal bovine serum, PDB, antibiotics). THP-1 cells were allowed to differentiate for 4 days prior to transfection with either pLNCX2-EGFP or pLNCX2-EGFP-RhoAT19N (1 µg of plasmid DNA:4 µl of Lipofectamine reagent). After 24 h, the medium was removed, cells were converted to foam cells, and cholesterol efflux was assayed as described above. Enhanced green fluorescent protein (EGFP) expression was visualized using fluorescence microscopy as an indication of the transfection efficiency and transgene expression in cells. EGFP expression was observed within 48 h posttransfection and continued 5 days posttransfection.

PPAR Phosphorylation—Duplicate wells of THP-1 macrophages were incubated for 24 h with atorvastatin in 5% LPDS. Cells were scraped into lysis buffer (1.25%, w/w, (octylphenoxy)polyethoxyethanol, 1.25%, w/v, sodium deoxycholate, 0.0125 M sodium phosphate, pH 7.2, 2 mM EDTA, and 100 units/ml aprotinin) containing the phosphatase inhibitors sodium vanadate (0.2 mM) and sodium fluoride (50 mM). Cells were disrupted with five passages through an 18-gauge needle, incubated for 30 min on ice, and then centrifuged for 30 min at 4 °C (10,000 rpm). Protein from the whole cell extract (600 µg at a concentration of 1 µg/µl in phosphate-buffered saline) was incubated with 30 µl of the PPAR Affinity Sorbent gel slurry (Cayman Chemical) for 16 h, 4 °C. Beads were precipitated by brief centrifugation, washed three times with phosphate-buffered saline prior to resuspension in SDS loading buffer, and boiled for 5 min. Proteins were separated by 12% PAGE and transferred electrophoretically onto polyvinylidene difluoride membranes. The membranes were blocked (16 h, 4 °C) with 5% (w/v) BSA and 0.1% Tween 20 in Tris-buffered saline, and incubated 16 h with an affinity purified anti-human PPAR antibody (WAK-Chemie, Steinbach, Germany) followed by a 1 h incubation with a peroxidase-conjugated anti-rabbit IgG antibody (Santa Cruz). PPAR was detected using BM chemiluminescence blotting substrate. Quantification analysis of the developed films was performed using an imaging densitometer (GS-700).

To determine the extent of serine phosphorylation of PPAR, aliquots of whole cell extract (500 µg), were immunoprecipitated with anti-PPAR antibody as above and separated by 12% PAGE. Proteins were transferred onto polyvinylidene difluoride membranes, blocked 16 h with commercial blocking buffer (1% w/v in Tris-buffered saline, Roche Diagnostics) and then incubated with 2 µg/ml (in 0.5% commercial blocking buffer) of a rabbit anti-phosphoserine polyclonal antibody (Chemicon, Temecula, CA) for 4 h. The membranes were then washed, incubated with a peroxidase-conjugated anti-rabbit IgG antibody, and the phosphoserine bands were quantified as described above.

Dual Luciferase Reporter Assay—The TK-LXRE3-luc construct containing three copies of the LXR response element upstream of a luciferase reporter and the TK promoter-Renilla luciferase construct pRL-TK, were a kind gift of D. Ory (Washington School of Medicine, St. Louis, MO). HepG2 cells were seeded in 35-mm wells (Falcon Scientific) overnight (50% confluent) and transiently cotransfected with 1 µg/ml TK-LXRE3-luc and 0.1 µg/ml pRL-TK using Lipofectamine reagents (Invitrogen) and the protocol described above. Following transfection (24 h) cells were refed LPDS and incubated with vehicle alone, atorvastatin (10 µM), or TO901317 (24 h). Cells were harvested and firefly and Renilla luciferase activities measured using the dual-luciferase reporter assay system (Promega). The relative luciferase activity was calculated by normalizing LXRE promoter-driven firefly luciferase activity to control Renilla activity. Data from all experiments are presented as the relative luciferase activity (mean ± S.E.) from two independent sets of experiments, each with duplicate measurements.

View larger version (42K): [in this window] [in a new window]   FIG. 2. The effect of statins on de novo cholesterol synthesis and cholesterol efflux in THP-1 macrophages. A, macrophages were preincubated for 19 h with increasing doses of atorvastatin (Atorva, 1–10 µM) or simvastatin (Simva, 10 µM), and the incorporation of radiolabeled acetate into cholesterol was measured. Values represent the mean ± S.E. of the pmol total cholesterol/mg of cell protein, expressed as percent of control where *, p < 0.05 compared with control (n = 3). B, cholesterol efflux was measured from cholesterol-loaded macrophages incubated with FAF-BSA alone or in the presence of apoAI (10 µg/ml) or HDL3, (100 µg/ml) following a 24-h preincubation with increasing doses of atorvastatin (1–10 µM). Values represent the mean ± S.E., expressed as a percent of cholesterol effluxed to acceptor alone where *, p < 0.05 as compared with efflux to FAF-BSA alone and , p < 0.05 as compared with acceptor alone (n = 4).

Statistical Analysis—All values are presented as means ± S.E. Statistical significance was determined by comparing means using an unpaired Student's t test, unless stated otherwise. A value of p < 0.05 was considered statistically significant.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Inhibition of HMG-Co A Reductase Enhances Macrophage Cholesterol Efflux—To demonstrate that atorvastatin inhibits de novo cholesterol synthesis in THP-1 macrophages, we measured the incorporation of radiolabeled acetate into cholesterol. Preincubation of THP-1 macrophages with increasing doses of atorvastatin (1–10 µM) for 19 h significantly reduced the incorporation of radiolabeled acetate into cholesterol by 60–87% (p < 0.05, Fig. 2A). In J774A.1 macrophages, atorvastatin similarly inhibited cholesterol synthesis with the maximal dose (10 µM) decreasing cholesterol synthesis up to 90% (data not shown).

Cholesterol efflux can be mediated by lipid-poor apolipoproteins such as apoAI via ABCA1, phospholipid-containing particles, such as HDL, and scavenger receptor B-1, and passive, receptor-independent, aqueous diffusion (27). Therefore, we determined whether cholesterol synthesis inhibition affected cholesterol efflux to medium supplemented with FAF-BSA alone or in the presence of apoAI or HDL₃. In macrophages exposed to atorvastatin (1–10 µM), cholesterol efflux to FAF-BSA alone was dose dependently enhanced from 1.07- to 1.7-fold (p < 0.05, Fig. 2B), compared with control. Addition of apoAI increased cholesterol efflux 2-fold (p < 0.05), which was further enhanced by atorvastatin, in a dose-dependent manner by 1.1–1.6-fold (p < 0.05, Fig. 2B) when compared with apoAI alone. Following correction for the amount of cholesterol effluxed to FAF-BSA alone, atorvastatin still enhanced cholesterol efflux to apoAI by up to 1.6-fold (p < 0.05, data not shown). Similarly, atorvastatin dose dependently enhanced cholesterol efflux to HDL₃ by 1.2–1.5-fold (p < 0.05, Fig. 2B). A related statin, simvastatin, was tested to demonstrate that the observed increase in cholesterol efflux was not unique to atorvastatin. Simvastatin enhanced cholesterol efflux to both apoAI and HDL₃ (1.55- and 1.25-fold, respectively, Fig. 2B), effects similar to those observed for atorvastatin. Both statins stimulated cholesterol efflux even though cellular cholesterol-loading by acetylated LDL, prior to efflux measurements, decreased cholesterol biosynthesis by 90% (data not shown).

View larger version (16K): [in this window] [in a new window]   FIG. 3. The effect of atorvastatin on the expression of cholesterol transporters in macrophages. THP-1 macrophages were incubated with atorvastatin (10 µM) for 24 h. ABCA1 and ABCG1 expression was determined as described under "Experimental Procedures," and the values represent the mean ± S.E. of the ratio of the band intensity of ABC relative to glyceraldehyde-3-phosphate dehydrogenase, expressed as a percent of control (n = 4). *, p < 0.05 relative to Me2SO control.

Exogenous Addition of Mevalonate Blocks the Atorvastatin-mediated Increase in Cholesterol Efflux—To demonstrate that atorvastatin enhances cholesterol efflux through inhibiting the synthesis of mevalonate and its metabolites, mevalonate was added to cells exogenously. Mevalonate alone had no effect on cholesterol efflux to either apoAI or HDL₃, as compared with control (Fig. 4A). However, the atorvastatin-mediated induction of cholesterol efflux to apoAI and HDL₃ (1.5- and 1.2-fold, respectively) was completely blocked by the addition of mevalonate (Fig. 4A). In addition to reversing the effects on cholesterol efflux, co-incubation of macrophages with exogenous mevalonate and atorvastatin blocked the statin-mediated induction in expression of ABCA1 (Fig. 4B) and ABCG1 (data not shown). Therefore, atorvastatin enhances cholesterol efflux and ABCA1 expression in part by inhibiting the synthesis of mevalonate and reducing subsequent downstream reactions.

Effect of Mevalonate Metabolites and Prenylation Inhibitors on Macrophage Cholesterol Efflux—In addition to depleting intracellular cholesterol levels, HMG-CoA reductase inhibitors also reduce the intracellular pool of non-sterol mevalonate metabolites such as FPP and GGPP (9). Therefore, we determined whether these isoprenoids played a role in mediating the increase in cholesterol efflux and ABCA1 expression by statins. Incubation of macrophages with FPP or GGPP alone, did not affect cholesterol efflux to apoAI or expression of ABCA1 (Fig. 5, A and C). However, co-incubation of macrophages with FPP in the presence of atorvastatin reversed the statin-mediated induction in cholesterol efflux to apoAI and ABCA1 mRNA expression. Similarly, GGPP completely blocked the increase in cholesterol efflux to apoAI and ABCA1 expression induced by atorvastatin (Fig. 5). These findings suggest that atorvastatin up-regulates cholesterol efflux in part through inhibiting isoprenoid synthesis.

Prenylation of proteins is catalyzed by either farnesyl transferases or geranylgeranyl transferases (19). Inhibition of farnesyl transferase inhibitor resulted in a significant increase in cholesterol efflux to apoAI (25%, p < 0.05) and similarly induced ABCA1 65% (Fig. 5, B and D) and ABCG1 expression (data not shown). In addition, an inhibitor of geranylgeranyl transferase, GGTI, significantly increased apoAI-mediated cholesterol efflux by 32% (p < 0.05) and markedly enhanced ABCA1 35% and ABCG1 expression by 30% (Fig. 5, B and D and data not shown). The increase in cholesterol efflux and ABCA1 expression observed with the inhibition of protein prenylation is consistent with the changes induced by statins, suggesting that farnesylated or geranylgeranylated proteins may be downstream mediators of the statin effect.

View larger version (24K): [in this window] [in a new window]   FIG. 4. The effect of mevalonate on the statin-mediated increase in ABC expression and cholesterol efflux in THP-1 macrophages. A, cholesterol efflux was measured from cholesterol-loaded THP-1 macrophages incubated with FAF-BSA alone or in the presence of apoAI (10 µg/ml) or HDL3, (100 µg/ml) following a 24-h preincubation with mevalonate (Meva, 100 µM) in the absence of presence of atorvastatin (Atorva, 10 µM). Values represent the mean ± S.E. of the percent cholesterol effluxed. *, p < 0.05 as compared with efflux to FAF-BSA alone; , p < 0.05 as compared with efflux to acceptor alone; , p < 0.05 as compared with efflux in the presence of acceptor plus atorvastatin (n = 3). B, ABCA1 mRNA expression was measured in THP-1 macrophages incubated in LPDS (5%) alone or with atorvastatin (10 µM), mevalonate (100 µM), or mevalonate and atorvastatin for 24 h, as described under "Experimental Procedures." Values represent the mean ± S.E. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

View larger version (36K): [in this window] [in a new window]   FIG. 5. The effect of isoprenoids and inhibitors of RhoA on cholesterol efflux and ABCA1 mRNA expression in THP-1 macrophages. A, cholesterol efflux was measured from cholesterol-loaded macrophages incubated with FAF-BSA alone or in the presence of apoAI (10 µg/ml) following a 24-h preincubation with either FPP or GGPP in the absence or presence of atorvastatin (Atorva, 10 µM). B, the effect of a farnesyl transferase inhibitor (FTI, 10 µM), GGTI (10 µM), and Rho protein inhibitor, C3 exoenzyme (10 µg/ml) on cholesterol efflux was also determined. Values represent the mean ± S.E. expressed as a percent of control (apoAI alone). *, p < 0.05 as compared with efflux to apoAI alone; , p < 0.05 as compared with efflux in the presence of atorvastatin (n = 3). C, ABCA1 mRNA expression was measured in THP-1 macrophages incubated in LPDS (5%) alone or with atorvastatin (10 µM), FPP (10 µM), GGPP (10 µM), FPP and atorvastatin, or GGPP and atorvastatin for 24 h as described under "Experimental Procedures." D, ABCA1 mRNA expression was determined in THP-1 macrophages incubated in LPDS alone or with farnesyl transferase inhibitor (FTI), GGTI, or C3 exoenzyme for 24 h. Values represent the mean ± S.E. of two separate experiments with triplicate measurements. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

To determine more directly a role for RhoA, THP-1 macrophages were transiently transfected with a bicistronic vector encoding EGFP and RhoA, containing the mutation T19N, known to confer a dominant-negative phenotype (43). Transfection efficiency ranged from 50–70% of the total number of cells, as determined by visualization of EGFP (Fig. 6C). Cholesterol efflux to apoAI in dominant-negative RhoA cells was increased 20% compared with those cells transfected with EGFP only (p < 0.05, Fig. 6D). These findings indicate a role for RhoA in the regulation of ABCA1-mediated cholesterol efflux.

One downstream target of RhoA is the serine/threonine kinase, ROCK (44). Addition of the specific inhibitor of ROCKI/II, Y27632 (45), to THP-1 cells increased ABCA1 mRNA expression (1.4-fold) and cholesterol efflux to apoAI (1.6-fold, p < 0.05).

Atorvastatin Mediates Its Effects through PPAR-LXR Activation—PPAR agonists induce ABCA1 expression through a molecular cascade involving LXR activation (3, 23, 24). Therefore we determined whether the atorvastatin-mediated increase in ABCA1 expression and, by extension, cholesterol efflux were dependent on the activation of PPAR and/or LXR. Initially, the effect of antagonists of PPAR and LXR on ABCA1 mRNA and cholesterol efflux was determined. The effectiveness of GW9662 and ECHS as PPAR and LXR antagonists, respectively, was established by demonstrating that these reagents blocked the ciglitizone (PPAR agonist)- and TO901317 (LXR agonist)-induced increase in cholesterol efflux and ABCA1 mRNA expression (Fig. 7). Importantly, co-incubation of GW9662 and atorvastatin inhibited the atorvastatin-induced increase in ABCA1 expression and apoAI-mediated cholesterol efflux by 50% (Fig. 7). Similar results were observed using another PPAR antagonist, bisphenol A diglycidyl ether (Fig. 7A) (39). Co-incubation of atorvastatin and the LXR antagonist, ECHS, completely blocked the statin-mediated induction of cholesterol efflux to apoAI. ECHS also blocked the statin-mediated increase in ABCA1 expression (Fig. 7).

PPAR activity can be negatively regulated through phosphorylation. Therefore, inhibition of RhoA activity by atorvastatin may result in reduced PPAR phosphorylation and enhanced PPAR activity. To test this hypothesis, we measured cellular levels of total PPAR protein and the extent of PPAR phosphorylation in the presence of atorvastatin by immunoblot analysis (Fig. 8, A and B). Total PPAR was unaffected by atorvastatin (5 and 10 µM) (Fig. 8A). To determine the level of phosphorylated PPAR in the presence of atorvastatin, cell lysates were immunoprecipitated with a PPAR-specific antibody, and serine phosphorylation was subsequently determined by immunoblot analysis using a specific phosphoserine polyclonal antibody. In atorvastatin-treated cells, the levels of phosphoserine residues were unchanged, indicating that the atorvastatin-mediated increase in PPAR activity was unrelated to decreased PPAR phosphorylation.

View larger version (36K): [in this window] [in a new window]   FIG. 6. RhoA cellular localization and the effect of dominant-negative RhoA on ABCA1-mediated cholesterol efflux. RhoA protein was measured in the cellular non-nuclear membranes and the cytosol isolated from THP-1 macrophages following incubation in the absence or presence of atorvastatin (Atorva) for 24 h, as described under "Experimental Procedures." A and B, representative immunoblot and quantification of RhoA expression in the cytosol and cellular membranes (n = 3). DMSO, Me2SO. C and D, THP-1 macrophages were transiently transfected with a bicistronic vector encoding EGFP and dominant-negative RhoA (T19N). The expression of EGFP (C) was visualized as an indicator of the efficiency of transfection and was detected using fluorescence microscopy (72 h posttransfection). The cholesterol efflux (D), in the absence (FAF-BSA) or presence of apoAI (10 µg/ml) was measured. The values represent the mean ± S.E. (n = 3). *, p < 0.05 as compared with efflux to FAF-BSA alone; , p < 0.05 as compared with efflux to apoAI in control cells (EGFP-transfected only).

Atorvastatin Reduces Macrophage Foam Cell Formation— Cellular CE deposition and therefore foam cell formation, is the net result of competing processes mediating lipoprotein uptake and cholesterol efflux. J774A.1 macrophages challenged with HTG-VLDL increased CE and triglyceride accumulation by 14- and 42-fold (p < 0.05, data not shown), respectively. Preincubation of macrophages with atorvastatin dose dependently inhibited CE accumulation induced by HTG-VLDL by 15–45% (Fig. 9). Atorvastatin did not significantly affect free cholesterol or triglyceride accumulation induced by HTG-VLDL (Fig. 9). Therefore, the atorvastatin-induced increase in cholesterol efflux contributes significantly to the inhibition of native lipoprotein-induced macrophage foam cell formation.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Mevalonate is the substrate for a sequence of reactions leading to the formation of the sterol and non-sterol metabolites, cholesterol and isoprenoids, respectively (9, 20). These metabolites can generate important signaling molecules; cholesterol is converted to oxysterols, and isoprenoids can posttranslationally modify and activate small GTPases (9, 20). Oxysterols regulate cholesterol homeostasis in part by modulating the expression of ABCA1 through LXR activation (46). Isoprenoid intermediates, which have long been associated with cell cycle control and cytoskeleton organization, have recently been shown to regulate LXR activation (10, 14). In this study we have established that statins enhance cholesterol efflux through a molecular cascade involving reduced isoprenoid synthesis, RhoA inactivation, LXR activation, and increased expression of ABCA1 and ABCG1. These findings further establish that non-sterol isoprenoids represent an additional endogenous pathway for the regulation of macrophage cholesterol homeostasis (10, 14).

HMG-CoA reductase inhibitors were used in this study to determine the consequences of cellular mevalonate depletion on macrophage cholesterol efflux. Treatment of cells with statins can result in the amplification of downstream regulatory pathways that are otherwise difficult to detect experimentally (47). Using this approach, we made the novel observation that incubation of cholesterol-loaded macrophages with atorvastatin or simvastatin significantly enhances cholesterol efflux to apoAI and HDL₃. This increase in cholesterol efflux was consistent with the statin-induced up-regulation of ABCA1 and ABCG1 expression. The effect of statins on both cholesterol efflux and ABC expression was completely blocked by co-incubation with mevalonate, implicating HMG-CoA reductase as the molecular target of inhibition. However, the increase in cholesterol efflux induced by statins was independent of their ability to inhibit the de novo synthesis of cholesterol. This was demonstrated by our observation that the cholesterol-loading phase of the efflux protocol resulted in substantial inhibition of newly synthesized cholesterol (80%), prior to the addition of the statin. Thus, statins appear to increase cholesterol efflux because of inhibition of the residual HMG-CoA reductase activity. HMG-CoA reductase is regulated by a complex multivalent feedback mechanism to ensure the synthesis of essential non-sterol metabolites continues, even in the presence of excess cholesterol (9). This regulation is accomplished by having HMG-CoA reductase activity sensitive to both sterol and non-sterol intermediates of the cholesterol biosynthetic pathway; however, the identity of the putative non-sterol intermediate is still uncertain (9, 48, 49). Therefore, when macrophage cholesterol synthesis is suppressed because of high levels of intracellular cholesterol, as in the efflux protocol, the small amount of mevalonate synthesized is preferentially diverted into the production of crucial non-sterol intermediates such as the isoprenoids (47). Our results demonstrate that statins enhance cholesterol efflux through inhibition of this branch of the mevalonate pathway.

View larger version (47K): [in this window] [in a new window]   FIG. 7. The role of PPAR and LXR in the statin-mediated increase in cholesterol efflux and ABCA1 mRNA expression. A, THP-1 macrophages were cholesterol-loaded, and the percent of cholesterol effluxed to apoAI (10 µg/ml) was measured following preincubation of macrophages with either ciglitizone (25 µM, PPAR agonist), TO901317 (1 µM, LXR agonist) or atorvastatin (Atorva, 10 µM), alone or in the presence of a PPAR antagonist (GW9662, 1 µM or bisphenol A diglycidyl ether, 10 µM) or an LXR antagonist (ECHS, 10 µM). Values represent the mean ± S.E., expressed as percent of control (n = 3 for all except for TO901317, where n = 2). *, p < 0.05 as compared with apoAI alone; , as compared with efflux in the presence of apoAI plus drug. B, ABCA1 mRNA was measured in THP-1 macrophages incubated with LPDS (5%) alone or with atorvastatin (10 µM) in the absence or presence of GW9662 (1 µM) or ECHS (10 µM). ABCA1 mRNA expression was also determined in cells incubated with either ciglitizone (25 µM) or TO901317 (1 µM) alone or in the absence or presence of GW9662 or ECHS for 24 h. Values represent the mean ± S.E. of two separate experiments with triplicate measurements. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

One consequence of decreased mevalonate synthesis by statins is a reduction in the isoprenoid intermediates, FPP and GGPP, which prenylate important signaling molecules such as the small GTP-binding proteins (20, 30). GTPases of the Ras and Rho family are dependent upon isoprenylation for proper membrane localization and function (20), a reaction inhibited by statins. Therefore, we explored the role of the specific mevalonate intermediates, FPP and GGPP, in modulating cholesterol efflux. Indeed, the addition of GGPP in the presence of atorvastatin significantly blocked the increase in ABCA1 mRNA expression and the enhancement in apoAI-mediated cholesterol efflux, whereas the inhibition of protein geranylgeranylation with GGTI mimicked the statin-induced increase in efflux. These data imply that a prenylated protein can modulate ABCA1 expression and cholesterol efflux. Three lines of experimental evidence implicated RhoA. First, incubation of macrophages with C3 exoenzyme, an enzyme that catalyzes ADP-ribosylation and renders Rho GTPases biologically inactive (12), significantly enhanced ABCA1 mRNA expression and cholesterol efflux. Second, statin treatment of macrophages significantly inhibited membrane localization of RhoA, a requirement for its activation. Third, the specific inactivation of RhoA through the transfection of macrophages with cDNA encoding dominant-negative RhoA significantly enhanced cholesterol efflux. Thus, regulation of RhoA activity modulates the extent of cellular cholesterol efflux.

View larger version (48K): [in this window] [in a new window]   FIG. 8. Effect of atorvastatin on PPAR phosphorylation and LXR-activated gene expression. Cellular lysates were prepared from THP-1 macrophages incubated with atorvastatin for 24 h. A, the amount of PPAR protein was determined by immunoblot (IB) analyses. PPAR was immunoprecipitated (IP) with an affinity-purified PPAR polyclonal antibody. PPAR was resolved on 12% PAGE. Total PPAR was determined by immunoblot analysis using the same polyclonal antibody. Band intensity values represent the density of PPAR from atorvastatin treated cells compared with Me2SO (DMSO) control. B, the amount of phosphorylated PPAR was determined following immunoprecipitation, resolved on 12% PAGE, and phosphorylated PPAR was detected with an anti-phosphoserine polyclonal antibody. Band intensities are expressed as relative densities compared with controls. C, LXR-activated gene expression was determined in HepG2 cells that were co-transfected with the TK-LXRE3-luc construct and the TK promoter-Renilla luciferase construct pRL-TK. Cells were incubated with Me2SO, 5 µM atorvastatin, 10 µM atorvastatin, or 1 µM TO901317 (24 h) and harvested, and the firefly and Renilla luciferase activities measured. The relative luciferase (rlu) activity was then calculated by normalizing LXRE promoter-driven firefly luciferase activity to control Renilla activity. Data from all experiments are presented as the relative luciferase activity (mean ± S.E.) from three independent sets of experiments. *, p < 0.05 as compared with control.

View larger version (36K): [in this window] [in a new window]   FIG. 9. The effect of atorvastatin on CE accumulation induced by atherogenic HTG-VLDL. J774A.1 macrophages were preincubated with atorvastatin (atorva, 1–10 µM) for 24 h prior to a 16-h incubation of atorvastatin in the absence or presence of HTG-VLDL (50 µg of lipoprotein cholesterol/mg of cell protein). CE, free cholesterol (FC), and triglyceride (TG) mass was determined as described under "Experimental Procedures." Values represent the mean ± S.E. expressed as percent of control (n = 3). *, p < 0.05 as compared with HTG-VLDL alone.

Therefore, we suggest that one consequence of statin-induced inhibition of RhoA and decreased activation of a Rho-associated kinase (19, 44) which contributes to LXR activation is an increase in PPAR activity. Our finding that approximately half of the statin-induced increase in cholesterol efflux is mediated through PPAR indicates that other mechanisms exist whereby decreased RhoA signaling activates LXR, and these remain to be determined. One Rho-associated kinase is the serine/threonine kinase, ROCK (44). Its involvement in the regulation of ABCAI was demonstrated by our observation that the specific inhibitor of ROCKI/II, Y27632 (45), increased ABCA1 mRNA expression and ABCA1-mediated cholesterol efflux to apoAI. The potential importance of the Rho/Rho-kinase pathway in atherogenesis has been demonstrated in animal models whereby treatment with inhibitors of Rho kinase reduced lesion development (53).

Our observation that prenylated proteins modulate cholesterol efflux through the regulation of LXR activation is entirely consistent with a recent study, which demonstrated that in THP-1 macrophages the addition of GGPP decreased ABCA1 expression, whereas GGTI and C3 exoenzyme increased ABCA1 expression (10). Furthermore, treatment of HepG2 cells with pitavastatin increased ABCA1 expression, a response dependent on the inhibition of HMG-CoA reductase and RhoA activities (54). Although cholesterol efflux was not measured in either study, Gan et al. (10) concluded that the down-regulation of ABCA1 by GGPP occurred through two mechanisms, directly by antagonism of LXR and indirectly through activation of RhoA (10). Consistent with these results, we observed that C3 exoenzyme increased ABCA1 expression and cholesterol efflux, similar to that observed with statin treatment. In our studies, GGPP alone did not decrease basal ABCA1 mRNA expression or cholesterol efflux; however, the addition of GGPP completely blocked the statin-induced increase in ABCA1 mRNA and cholesterol efflux.

Interestingly, we demonstrate that FPP completely blocks the statin-mediated increase in cholesterol efflux and that inhibition of farnesylation mimicked the statin effect. It is possible that GTPases, such as Ras, which are preferentially farnesylated, participate directly in the statin-mediated increase in cholesterol efflux. However, the addition of exogenous FPP may simply have provided more substrate for GGPP formation and that farnesyl transferase inhibitor shunted accumulated FPP into the GGPP synthesis pathway (20). Treatment of human monocytes with fluvastatin has been shown to reduce the functional activity of Ras (55), and in turn, regulate PPAR activity (56). Therefore, it is plausible that statins mediate enhanced LXR activation leading to increased cholesterol efflux, in part, through a Ras-induced increase in PPAR activity. Alternatively, Ras and Rho GTPases, through cross-talk or signal convergence may coordinate LXR activation, regulation of ABCA1 expression, and cholesterol efflux (19).

Increasing evidence suggests that Rho proteins play an important role in cardiovascular disease through modulating vascular cell responses including inflammation, oxidative stress, growth and endothelial nitric-oxide synthase expression (57). The realization that statins can inhibit Rho protein activity through decreasing isoprenoid synthesis has led to the elucidation of statin-induced modulation of vascular cell function (57). In the present study we revealed that modulation of RhoA activity can influence ABCA1 expression, macrophage cholesterol efflux, and the induction of foam cell formation by atherogenic lipoproteins.

	FOOTNOTES

 
^* This work was supported by Grant MT 8014 (to M. W. H.) and a studentship from the Canadian Institutes for Health Research (to C. A. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^|| Career Investigators of the Heart and Stroke Foundation of Ontario.

^** Holds a Canada Research Chair (Tier I) in Human Genetics.

To whom correspondence should be addressed: Robarts Research Institute, Rm. 4-16, 100 Perth Dr., London, Ontario, Canada, N6A 5K8. Tel.: 519-663-3793; Fax: 519-663-3112; E-mail: mhuff{at}uwo.ca.

¹ The abbreviations used are: PPAR, peroxisome proliferator activated receptor; LXR, liver X receptor; HMG, 3-hydroxy-3-methylglutaryl; FPP, farnesyl pyrophosphate; GGPP, geranylgeranyl pyrophosphate; SREBP, sterol regulatory element-binding protein; SCAP, SREBP cleavage activating protein; ABC, ATP binding cassette; HDL, high density lipoprotein; CE, cholesteryl ester; LDL, low density lipoprotein; HTG-VLDL, hypertriglyceridemic very low density lipoproteins; BSA, bovine serum albumin; GGTI, geranylgeranyl transferase inhibitor; ECHS, 5,6-epoxycholesterol-3-sulfate; EGFP, enhanced green fluorescent protein; FAF, fatty acid-free; LPDS, lipoprotein-deficient serum; LXRE, liver x receptor response element.

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