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CARDIOVASCULAR

Pitavastatin Effect on ATP Binding Cassette A1-Mediated Lipid Efflux from Macrophages: Evidence for Liver X Receptor (LXR)-Dependent and LXR-Independent Mechanisms of Activation by cAMP

Department of Pharmacological and Biological Sciences and Applied Chemistries, University of Parma, Parma, Italy (I.Z., F.P., E.F., F.B.); and Department of Biosciences, Karolinska Institutet Novum, Huddinge, Sweden (K.R.S., J.-Å.G.)

Received August 6, 2005; accepted January 11, 2006.

	Abstract

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The promotion of lipid efflux from macrophages is an important ATP binding cassette A1 (ABCA1)-mediated antiatherosclerotic mechanism that prevents peripheral tissues from foam cell accumulation. Statins exert beneficial antiatherosclerotic effects on cardiovascular disease correlated to the cholesterol-lowering properties and the pleiotropic activities. In this work, we investigated the ability of statins to modulate ABCA1-mediated lipid efflux from macrophages, where the protein expression was differently induced. Pitavastatin (0.1–10 µM) and compactin (10 µM) reduced both cholesterol and phospholipid efflux up to 60% from macrophages expressing ABCA1 upon treatment with 8-(4-chlorophenylthio)-cyclic AMP (cpt-cAMP), and this was secondary to a reduction of ABCA1 mRNA and protein content. Conversely, statins did not affect ABCA1 activity when the protein was up-regulated by 22-hydroxycholesterol/9-cis-retinoic acid or through cholesterol loading. Statin inhibition of lipid efflux induced by cpt-cAMP was reversed in the presence of mevalonate, 22-hydroxycholesterol, and cholesterol but not geranyl geraniol. In macrophages obtained from liver X receptor (LXR)-deficient mice, cpt-cAMP still promoted cholesterol efflux, but pitavastatin did not exert any effect. The present work shows that statins may inhibit ABCA1-mediated lipid efflux in macrophages only when ABCA1 protein expression is induced by cpt-cAMP and provides evidence that cAMP may activate ABCA1 independently of an increase of intracellular sterol synthesis but through at least two pathways: one independent of LXR and one involving an intracellular sterol(s) acting as LXR ligand(s). In addition, the lack of inhibitory effect on lipid efflux in cholesterol-loaded macrophages is likely to exclude a potential negative pleiotropic effect by statins.

ABCA1 is a ubiquitously distributed protein, whose expression is regulated both at transcriptional and post-translational levels. An influence on gene transcription has been shown after activation of the nuclear receptors peroxisome proliferator-activated receptor (Chinetti et al., 2001), liver X receptor (LXR) and retinoid X receptor (RXR) (Costet et al., 2000), treatment with cytokines and inflammatory agents (Wang et al., 2002; Khovidhunkit et al., 2003), and enrichment of macrophages with cholesterol (Langmann et al., 1999) or cAMP analogs (Bortnick et al., 2000). Stimulation of the LXR-RXR system occurs through the binding of specific ligands, such as oxysterols and 9-cis-retinoic (cRA) acid, respectively, to the receptors, leading to the formation of the heterodimer LXR/RXR and its binding to a promoter sequence on the ABCA1 gene (Costet et al., 2000; Singaraja et al., 2001). Likewise, cholesterol enrichment of cells seems to promote ABCA1 transcription by the same mechanism: even if cholesterol itself is not a direct ligand of LXR, its conversion into oxidative derivatives results in the up-regulation of the transporter (Langmann et al., 1999). The molecular pathway of cAMP-mediated stimulation of ABCA1 expression has not been fully elucidated yet. The proposed mechanism for the regulation of ABCA1 in response to cAMP involves a cAMP response element-binding protein that would interact with a specific promoter region of the gene (Srivastava, 2002). Alternatively, cAMP could mediate the phosphorylation of a Sp1 factor at the ABCA1 promoter (Schmitz and Langmann, 2005). In addition, some studies demonstrated that cAMP may also potentiate ABCA1 activity through phosphorylation of the protein that stabilizes it (Haidar et al., 2002). Other factors positively or negatively affect function and stabilization of ABCA1 protein; apoprotein A-I (apoA-I) and free cholesterol have been demonstrated, respectively, to increase (Arakawa et al., 2004) and reduce (Feng and Tabas, 2002) ABCA1 protein content. Pharmacologically, probucol impairs ABCA1 transporter activity by blocking its translocation from late endosomes to the cellular membrane (Favari et al., 2004).

Recently, several reports have revealed that treatment with 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) may modulate ABCA1 expression in vitro (Ando et al., 2004; Sone et al., 2004; Wong et al., 2004; Argmann et al., 2005). However, data reported in the literature on the effects of statins on ABCA1 activity in macrophages are quite variable; some authors found down-regulation of mRNA and protein followed by a decrease in the cholesterol release to apoA-I (Wong et al., 2004). Others found a stimulatory effect of atorvastatin on gene expression that translated into a potentiated lipid efflux in the same cell model (Argmann et al., 2005). We showed that treatment of rat hepatoma Fu5AH with statins resulted in an improvement of ABCA1-mediated release of cholesterol and phospholipids (Zanotti et al., 2004).

Statins represent the first-line agents for lipid lowering in patients with atherosclerosis and cardiovascular disease (Kjekshus et al., 1996; Rosenson and Tangney, 1998), whose cellular mechanism of action is attributed to the inhibition of cellular cholesterol synthesis in the liver (Goldstein and Brown, 1990). Blocking of the mevalonate pathway depletes cells not only of cholesterol but also of numerous metabolites involved in different cell functions; thus, statin activity has the potential to result in pleiotropic effects consisting of modification of endothelial function, reduction of inflammatory responses, and improvement in plaque stability (Bernini et al., 1995; Corsini et al., 1999). The newly discovered effect on ABCA1 may be recognized as a pleiotropic effect of potential interest, given the correlation of ABCA1 with clinical events (Clee et al., 2000; van Dam et al., 2002; Singaraja et al., 2003) and the importance of statins as pharmacological agents in therapeutic use. In the present work, we investigated the ability of pitavastatin, a newly synthesized HMG-CoA reductase inhibitor (Mukhtar et al., 2005), to modulate ABCA1-mediated cholesterol efflux from macrophages, the key cells in atherosclerosis, where ABCA1 expression was differently induced.

	Materials and Methods

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Chemicals. Pitavastatin was kindly provided by Kowa Company Ltd. (Tokyo, Japan). Organic solvents were purchased from Merck (Darmstadt, Germany). 8-(4-Chlorophenylthio)-cyclic AMP (cpt-cAMP), cRA, 22-hydroxycholesterol (22OH), compactin, mevalonate, and geranyl geraniol (GGOH) were purchased from Sigma Chemical Co. (St. Louis, MO). [1,2-³H]Cholesterol, [methyl-³H]choline chloride, and [2-¹⁴C]acetate sodium salt were from Amersham Biosciences (Uppsala, Sweden). Western blot buffers and supplies were purchased from Invitrogen (Carlsbad, CA). Acetylated low density lipoprotein (AcLDL) was prepared from human LDL as described previously (Bernini et al., 1997). ApoA-I was kindly donated by Dr. Laura Calabresi (University of Milan, Milan, Italy).

Cells. Mouse peritoneal macrophages (MPMs) were collected from the peritoneum of thioglycollate-treated wild-type mice (Charles River, Calco, Italy) or LXR- and -deficient mice generated as described previously (Alberti et al., 2001) and cultured in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS) (both from Cambrex Bioscience, Verviers, Belgium) and gentamicin (Invitrogen). J774 macrophages were cultured in RPMI 1640 and antibiotics. All cells were seeded in 24-well plates for efflux experiments and incubated at 37°C and 5% CO₂.

Cholesterol Efflux. Cells were labeled with 2 µCi/ml [1,2-³H]-cholesterol in RPMI 1640 + 1% FCS with or without 50 µg/ml AcLDL for 24 h; successively, cells were equilibrated for 18 h in RPMI 1640 + 0.2% bovine serum albumin (BSA) in the presence or absence of statins. During this period, ABCA1 might be induced by treatment with 0.3 mM cpt-cAMP or an association of 5 µg/ml 22OH and 10 µM cRA. Cholesterol efflux was promoted for 4 to 6 h to apoA-I in RPMI 1640, and the radioactivity present in the medium was determined by liquid scintillation counting. To analyze cellular [³H]cholesterol content, cell monolayers were extracted by the addition of 0.6 ml of 2-propanol. The lipid extracts were dried under a stream of N₂, resuspended in toluene, and quantified by liquid scintillation counting.

Phospholipid Efflux. The phospholipid efflux was performed as described previously (Yancey et al., 1995). After the labeling period of 48 h with 4 µCi/ml [methyl-³H]choline chloride in RPMI 1640 + 1% FCS with or without 50 µg/ml AcLDL, cells were equilibrated for 18 h in RPMI 1640 + 0.2% BSA in the presence or absence of statins. In this equilibration period ABCA1 was induced differently by treatment with 0.3 mM cpt-cAMP or an association of 5 µg/ml 22OH and 10 µM cRA. Efflux was promoted to 20 µg/ml apoA-I for 6 h in RPMI 1640. At that time point, media were centrifuged, and the supernatant was removed and extracted by the Bligh and Dyer method (Iverson et al., 2001).

Western Blotting Analysis. Cell monolayers were lysed in a 1% Triton X-100, 0.5% Nonidet P-40, and 10 mM Tris buffer and homogenized through a 27-gauge needle. Equal amounts of protein (40 µg) were separated on 5% tris acetate gels and transferred to a polyvinylidene fluoride membrane. ABCA1 was detected with a rabbit primary antibody (Novus Biological, Cambridge, UK) and a secondary antibody, anti-rabbit IgG conjugated to horseradish peroxidase, with visualization by enhanced chemiluminescence (ECL Plus) (both from GE Healthcare, Little Chalfont, Buckinghamshire, UK) according to the manufacturer's conditions.

RNA Extraction and Real-Time Quantitative PCR. The isolation of total RNA was achieved using the NucleoSpin RNA II according to the manufacturer's instructions (Macherey-Nagel, Duren, Germany). Reverse transcription was done using a cDNA archive kit (Applied Biosystems, Foster City, CA). The resulting cDNA was used for the real-time quantitative PCR in the ABI Prism 7000 sequence detection system (Applied Biosystems). The specific primers and TaqMan probes for murine ABCA1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were obtained from Applied Biosystems (Assays-on-Demand Gene Expression Products and TaqMan Rodent GAPDH Control Reagents). To control for the variation in the amount of DNA available for PCR in the different samples, gene expression of the target sequence was normalized in relation to the expression of an endogenous control, GAPDH.

Synthesis of Total Sterols. The synthesis of cholesterol was determined by measuring the incorporation of radioactive acetate into total cellular sterols (Brown et al., 1978). After incubation with [2-¹⁴C]acetate (2 µCi/ml) for 20 h, cell monolayers were washed with phosphate-buffered saline and digested with 0.1 mM NaOH. Aliquots were saponified at 60°C for 1 h in alcoholic KOH after the addition of [1,2-(n)-³H]cholesterol as an internal standard (10⁵ cpm/sample). The unsaponifiable material was extracted with low-boiling point petrol ether and counted for radioactivity. To evaluate the incorporation of labeled acetate into cellular sterols, these were separated from the unsaponifiable fraction by thin-layer chromatography by using petroleum ether (boiling point, 40–60°C)/diethyl ether/acetic acid (70:30:1). Radioactivity was measured by liquid scintillation counting.

Statistical Analysis. Results are reported as means of triplicates ± S.D. Statistical significance was determined by two-tailed Student's t test.

	Results

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As previously shown, ABCA1 expression can be up-regulated in cell cultures of macrophages by three different approaches: 1) treatment with analogs of cAMP (Bortnick et al., 2000); 2) treatment with ligands of nuclear receptors LXR and RXR (Costet et al., 2000); and 3) cholesterol loading of cells (Costet et al., 2000). First, we investigated the effect of pitavastatin and compactin on ABCA1-mediated cholesterol release from macrophages preincubated with cpt-cAMP. Treatment of J774 macrophages with both statins resulted in a 60% reduction of cholesterol efflux (Fig. 1). The pitavastatin inhibitory effect was maximum at concentrations between 0.1 and 10 µM. The latter concentration was chosen for all of the following experiments. To rule out the possibility that the statin inhibition of cholesterol efflux observed in our study only involved a depletion of substrate, we evaluated pitavastatin and compactin effects on phospholipid efflux, another specifically ABCA1-mediated process. ABCA1 was induced in J774 macrophages by 0.3 mM cpt-cAMP, and lipid efflux was promoted to apoA-I. Again, pitavastatin and compactin treatments produced a significant decrease in the amount of radioactivity released into the cell medium (Fig. 2), suggesting that ABCA1 activity is specifically impaired by statins.

View larger version (12K): [in this window] [in a new window]   Fig. 1. Effect of statins on cholesterol efflux to apoA-I from J774 macrophages pretreated or not with cpt-cAMP. Cells were radiolabeled with 2 µCi/ml [3H]cholesterol for 24 h in RPMI 1640 + 1% FCS followed by an equilibration period of 18 h in RPMI 1640 + 0.2% BSA in the presence or absence of 0.1 to 10 µM pitavastatin or 10 µM compactin. In pretreated cells, 0.3 mM cpt-cAMP was added during the 18-h equilibration period. Efflux was promoted to 20 µg/ml apoA-I for 4 h in RPMI 1640 and evaluated as counts per minute in medium/(counts per minute in medium + counts per minute in monolayers) x 100. Data are expressed as means ± S.D. (n = 3). *, p < 0.05; **, p < 0.01 compared with control.

View larger version (10K): [in this window] [in a new window]   Fig. 2. Effect of statins on phospholipid efflux to apoA-I from macrophages pretreated or not with cpt-cAMP. Cells were radiolabeled with 4 µCi/ml [3H]choline for 48 h in RPMI 1640 + 1% FCS followed by an equilibration period of 18 h in RPMI 1640 + 0.2% BSA in the presence or absence of 10 µM pitavastatin or 10 µM compactin. In pretreated cells, 0.3 mM cpt-cAMP was added during the 18-h equilibration period. Efflux was promoted to 20 µg/ml apoA-I for 6 h in RPMI 1640 and evaluated as counts per minute in medium/(counts per minute in medium + counts per minute in monolayers) x 100. Data are expressed as means ± S.D. (n = 3). **, p < 0.01 compared with control.

View larger version (15K): [in this window] [in a new window]   Fig. 3. Effect of pitavastatin on cholesterol (top) and phospholipid (bottom) efflux to apoA-I from J774 macrophages treated with 22OH/cRA or AcLDL. Cells were radiolabeled with 2 µCi/ml [3H]cholesterol for 24 h (top) or 4 µCi/ml [3H]choline for 48 h (bottom) in RPMI 1640 + 1% FCS followed by an equilibration period of 18 h in RPMI 1640 + 0.2% BSA in the presence or absence of 10 µM pitavastatin. ABCA1 was up-regulated by incubation with 50 µg/ml AcLDL in the labeling periods or by incubation with 5 µg/ml 22OH and 10 µM cRA in the 18-h equilibration period. Efflux was promoted to 20 mg/ml apoA-I for 4 (top) or 6 h (bottom) in RPMI 1640 and expressed as counts per minute in medium/(counts per minute in medium + counts per minute in monolayers) x 100. Data are expressed as means ± S.D. (n = 3). *, p < 0.05 compared with control.

The ability of statins to differently modulate cholesterol efflux was confirmed in MPMs. Whereas both pitavastatin and compactin significantly reduced lipid release when ABCA1 was up-regulated by cpt-cAMP, incubation with AcLDL or 22OH-cRA prevented this effect (Table 1).

Statin inhibition of ABCA1 activity under specific experimental conditions allowed us to hypothesize that these molecules may interfere with some steps in the molecular pathway of ABCA1 activation promoted by cpt-cAMP. Although the discovery that cAMP is an ABCA1 activator by a transcriptional mechanism, a sequence on the ABCA1 promoter specifically recognized by cAMP has not been definitively identified (Kiss et al., 2005).

The statin mechanism of action was further investigated by the addition of mevalonate, GGOH, or 22OH in J774 macrophages treated with pitavastatin and cpt-cAMP. In fact, if the inhibitory effect of statins on cholesterol efflux was related to the pharmacological activity of statins on HMG-CoA reductase, we could expect that exogenous supplementation of a cholesterol precursor or an intermediate in the cholesterol biosynthetic pathway downstream of HMG-CoA reductase could reconstitute normal cholesterol efflux. Moreover, because we have demonstrated that statins do not exert inhibitory effects in macrophages loaded with 22OH, we hypothesized that the high degree of inhibition in cpt-cAMP-treated cells could be attenuated in macrophages treated with the oxysterol. Although the intermediate of cholesterol synthesis, mevalonate, completely restored the control cholesterol efflux, GGOH did not exert any significant effect compared with cells treated with pitavastatin (Fig. 4), suggesting that depletion of nonisoprenoid derivatives of cholesterol is involved in statin-mediated inhibition. Moreover, the inhibition of cholesterol efflux to apoA-I completely disappeared in the presence of 22OH (Fig. 4), indicating that the depletion of a sterol derivative of cholesterol may be involved in the mechanism of action of pitavastatin inhibition of cholesterol efflux. Consistent with the observation that an exogenous supplementation of cholesterol reduces the inhibitory effect of statins on cAMP-mediated efflux, J774 macrophages loaded with AcLDL and successively equilibrated in the presence of cpt-cAMP and pitavastatin maintained an intact capacity to efflux cholesterol (data not shown).

View larger version (28K): [in this window] [in a new window]   Fig. 4. Effect of mevalonate, GGOH, and 22OH on pitavastatin-induced inhibition of cholesterol efflux from J774 macrophages pretreated with cpt-cAMP. Cells were radiolabeled with 2 µCi/ml [3H]cholesterol for 24 h in RPMI 1640 + 1% FCS followed by an equilibration period of 18 h in RPMI 1640 + 0.2% BSA and 0.3 mM cpt-cAMP in the presence or absence of 10 µM pitavastatin. During the 18-h pharmacological treatment with pitavastatin, 100 µM mevalonate, 10 µM GGOH, or 5 µg/ml 22OH was added to the cells. Efflux was promoted to 20 µg/ml apoA-I for 4 h and expressed as counts per minute in medium/(counts per minute in medium + counts per minute in monolayers) x 100. Data are expressed as means ± S.D. (n = 3). *, p < 0.05 compared with cpt-cAMP; +, p < 0.05 compared with pitavastatin.

Because cAMP-dependent ABCA1 activity is highly impaired by statins, it is conceivable that gene and/or protein expression in macrophages could be altered upon treatment with pitavastatin. Recent reports showed that statins may impair ABCA1 expression in macrophagic cells (Ando et al., 2004; Sone et al., 2004; Wong et al., 2004), but the potential interference with cAMP-mediated stimulation has not been investigated. As expected, pitavastatin significantly reduced ABCA1 mRNA and protein content in J774 macrophages incubated with cpt-cAMP; fully consistent with the functional data, 100 µM mevalonate, but not 10 µM GGOH, eliminated this inhibition (Fig. 5, A and B).

View larger version (46K): [in this window] [in a new window]   Fig. 5. Effect of pitavastatin, mevalonate, and GGOH on ABCA1 expression in J774 macrophages pretreated with cpt-cAMP. Cells were incubated in RPMI 1640 + 0.2% BSA with or without 0.3 mM cpt-cAMP. Simultaneously, 10 µM pitavastatin, 100 µM mevalonate, or 10 µM GGOH was added where indicated. A, mRNA was isolated from cells and converted into cDNA. The mRNA expression level of ABCA1 gene was assayed by reverse transcription-PCR and normalized with GAPDH mRNA level. Data are means of triplicates ± S.D. *, p < 0.05 compared with cpt-cAMP; +, p < 0.05 compared with pitavastatin. B, protein was isolated as described under Materials and Methods; ABCA1 was detected with a rabbit primary antibody to ABCA1 and a secondary rabbit antibody conjugated to horseradish peroxidase.

To support this hypothesis, the ability of cpt-cAMP to stimulate ABCA1-mediated cholesterol efflux and the statin effect was tested in MPMs from LXR-deficient mice. Interestingly, cells that lacked LXR maintained the ability to efflux cholesterol upon treatment with cpt-cAMP, but in these cells, pitavastatin did not affect the process (Fig. 6). To verify whether cpt-cAMP stimulation of ABCA1 could involve an up-regulation of intracellular sterol synthesis, we evaluated the incorporation of [2-¹⁴C]acetate into total cellular sterols. Data in Table 2 indicate that cpt-cAMP did not modify or even reduce sterol synthesis, whereas, as expected, pitavastatin strongly inhibited this cellular process.

View larger version (25K): [in this window] [in a new window]   Fig. 6. Effect of cpt-cAMP and pitavastatin (pita) on ABCA1-mediated efflux to apoA-I from wild-type (WT) or LXR-deficient MPMs. Cells were radiolabeled with 2 µCi/ml [3H]cholesterol for 24 h in RPMI 1640 + 1% FCS followed by an equilibration period of 18 h in RPMI 1640 + 0.2% BSA in the presence or absence of 10 µM pitavastatin. Where indicated, ABCA1 was up-regulated by 0.3 mM cpt-cAMP added during the 18-h equilibration period. Efflux was promoted to 5 µg/ml apoA-I for 6 h in RPMI 1640 and expressed as counts per minute in medium/(counts per minute in medium + counts per minute in monolayers) x 100. Data are expressed as means ± S.D. (n = 3). *, p < 0.05 compared with basal; ++, p < 0.01 compared with cpt-cAMP.

	Discussion

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Removal of excess cholesterol from arterial wall by the ABCA1 transporter is associated with protection from coronary artery disease (Clee et al., 2000; van Dam et al., 2002), and ABCA1 hepatic activity promotes the formation of high-density lipoprotein, the key lipoprotein in reverse cholesterol transport. Therefore, therapies designed to promote cholesterol efflux might have substantial therapeutic benefit. We recently reported that statins used worldwide for the treatment of cardiovascular disease significantly increase ABCA1-mediated lipid release from a model of hepatic cells, providing evidence of a new, beneficial pleiotropic effect of this class of compounds (Zanotti et al., 2004). In the present work, we investigated the effect of pitavastatin on ABCA1 activity in macrophages, the most important cholesterol-accumulating cells in atherosclerosis. We reported that HMG-CoA reductase inhibition reduces ABCA1-mediated lipid efflux in J774 macrophages specifically when this transporter is induced by cpt-cAMP, whereas no effect is detected when stimulation is achieved by incubation of cells with ligands of LXR and RXR or cellular enrichment with cholesterol. The inhibitory effect of cholesterol efflux by pitavastatin observed in J774 macrophages pretreated with cpt-cAMP was in parallel with a decrease of the cellular content of ABCA1 mRNA and protein. Two main mechanisms have so far been suggested for ABCA1 activation by cAMP; one occurs through the increase in ABCA1 mRNA (Bortnick et al., 2000), and the other one involves the post-translational stabilization of the ABCA1 protein through a PKA-dependent pathway (Haidar et al., 2002). Our results showing that statins may exert an inhibitory effect on cAMP-mediated activation of gene transcription support the validity of the first hypothesis and are consistent with a recent study indicating that, in J774 macrophages, cAMP stimulates ABCA1 activity preferentially by the promotion of gene transcription rather than through a phosphorylation pathway (Kiss et al., 2005).

Nevertheless, the exact mechanism by which cAMP increases the ABCA1 level is still under investigation; previous studies indicated that cAMP signaling may not require intracellular sterols (Schmitz and Langmann, 2001), although direct evidence of LXR independence has not been provided. Our results clearly indicate that pitavastatin inhibition of the mevalonate pathway interferes with the cAMP signaling that leads to ABCA1 up-regulation, suggesting that cAMP may require a cholesterol derivative or an intermediate of cholesterol biosynthesis to induce ABCA1. In fact, the inhibition by statins of cAMP-induced ABCA1-mediated efflux is reversed by mevalonate, oxysterol, or cholesterol and not by geranyl geraniol. These results are consistent with the observation that downstream products of mevalonate are involved in LXR activation in cultured cells (Forman et al., 1997). We suggest that cellular sensitivity to statin treatment under our experimental conditions is attributable to the ability of cAMP to induce ABCA1 expression through a sterol-activated LXR pathway. This conclusion is supported by our observation that pitavastatin does not inhibit cholesterol efflux induced by cAMP in LXR-deficient MPM. An interaction of cAMP and the LXR pathway was already documented in a recent work, indicating that LXR can bind to a cAMP response element and function as a transcription factor of renin gene (Morello et al., 2005). In cells not treated with cpt-cAMP, lipid efflux to apoA-I was very low. In these conditions, statins exert a modest inhibitory effect on cholesterol efflux. The low rate of efflux observed in basal conditions is most probably sustained by LXR stimulation because of the intracellular synthesis of sterols (Forman et al., 1997); therefore, a certain inhibitory effect of statins could be expected, even in the absence of cAMP stimulation. However, in the presence of cAMP, statins were more active in inhibiting ABCA1-mediated lipid efflux, despite the fact that the cholesterol synthesis in this condition was similar to or even lower than that in the basal condition. This observation excludes the possibility that the ability of cAMP to stimulate ABCA1 activity may involve an increase of sterol synthesis, which in turn might result from stimulation of cholesterol efflux itself and supports the specificity of statin action on the cAMP-mediated induction of ABCA1.

The recently reported results concerning statin effects on ABCA1 are very controversial; these compounds have been shown either to enhance or reduce the transporter expression and activity depending on the cell line (Ando et al., 2004; Sone et al., 2004). Furthermore, even with the same THP-1 cell model, authors have described either a down-regulation (Wong et al., 2004) or an up-regulation of cholesterol efflux (Argmann et al., 2005). These varying results may be related to the dual effect of mevalonate derivatives on ABCA1 activation. Statin-induced depletion of cellular 24,25-epoxycholesterol, recognized as a potent LXR activator, mainly resulted in reduction of ABCA1 activation via LXR (Wong et al., 2004). On the other hand, depletion of isoprenoid products of mevalonate synthesis by statins led to the suppression of an inhibitory pathway of ABCA1 (Argmann et al., 2005). The latter mechanism was proposed by us to explain the observation that pitavastatin dose-dependently increased ABCA1 release of cholesterol and phospholipids from Fu5AH rat hepatoma cells (Zanotti et al., 2004).

Therefore, it is clear that statin inhibition of HMG-CoA reductase may induce an opposite influence on ABCA1, and the final effect is not obvious, depending on the intracellular balance between the isoprenoid and nonisoprenoid branch of the mevalonate pathway. Under our experimental conditions, isoprenoid products are not likely to be involved in the statin effect, because the addition of GGOH to cells treated with pitavastatin did not reconstitute normal efflux. We suggest that, in the cpt-cAMP-stimulated macrophages, the effect of pitavastatin on depletion of intermediates of cholesterol biosynthetic pathway prevails over the reduction of isoprenoid products, with a resulting overall inhibitory effect on LXR activation and ABCA1 activity.

Our present results on macrophages expressing ABCA1 upon incubation with cpt-cAMP indicate that, in these cells, at least two regulatory pathways are operative. 1) One is sensitive to statins, is mediated by an endogenous sterol acting as an LXR agonist, and does not involve isoprenoid products, and 2) the second one is statin-insensitive and does not involve either the LXR or the mevalonate pathway.

Finally, the fact that the inhibitory activity of statins on ABCA1 does not occur in cholesterol-loaded macrophages suggests that this pleiotropic effect will not negatively influence the antiatherogenic effects of statins. This observation is of particular interest, considering the wide use of statins in clinical practice.

	Acknowledgements

 
We thank Prof. George H. Rothblat and Dr. Paolo Parini for helpful discussion, Dr. Nicola Ferri for excellent assistance in performing reverse transcription-PCR, and Dr. Laura Calabresi for providing apoA-I.

	Footnotes

 
The work was supported by Grants from Istituto Nazionale per le Ricerche Cardiovascolari, Compagnia di San Paolo and the Italian Ministry of University and Scientific Research and by a grant from Kowa Co. LTD. F.B. is also supported by the United States-Italy Scientific Exchange Agreement.

This work was presented in part at the 75th European Atherosclerosis Society Congress; 2005 April 23–26, Prague, Czechoslovakia.

doi:10.1124/jpet.105.093930.

ABBREVIATIONS: ABCA1, ATP binding cassette A1; LXR, liver X receptor; RXR, retinoid X receptor; cRA, 9-cis-retinoic acid; apoA-I, apoprotein A-I; HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; cpt-cAMP, 8-(4-chlorophenylthio)-cyclic AMP; 22OH, 22-hydroxycholesterol; GGOH, geranyl geraniol; AcLDL, acetylated low-density lipoprotein; MPM, mouse peritoneal macrophage; FCS, fetal calf serum; BSA, bovine serum albumin; PCR, polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Address correspondence to: Prof. Franco Bernini, Department of Pharmacological and Biological Sciences and Applied Chemistries, School of Pharmacy, University of Parma, viale delle Scienze 27/A, 43100 Parma, Italy. E-mail: fbernini{at}unipr.it

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