Liver X Receptor Stimulates Cholesterol Efflux and Inhibits Expression of Proinflammatory Mediators in Human Airway Smooth Muscle Cells

Abstract

Human (h) airway smooth muscle (ASM) cells are important mediators of the inflammatory process observed in asthma and other respiratory diseases. We show here that primary hASM cells express liver X receptor (LXR; α and β subtypes), an oxysterol-activated nuclear receptor that controls expression of genes involved in lipid and cholesterol homeostasis, and inflammation. LXR was functional as determined by transient assays using LXR-responsive reporter genes and by analysis of mRNA and protein expression of endogenous LXR target genes in cells exposed to LXR agonists. LXR activation induced expression of the ATP-binding cassette transporters ABCA1 and ABCG1 and increased efflux of cholesterol to apolipoprotein AI and high-density lipoprotein acceptors, pointing to a role for hASM cells in modulating cholesterol homeostasis in the airway. Under inflammatory conditions, hASM cells release a variety of chemokines and cytokines that contribute to inflammatory airway diseases. Activation of LXR inhibited the expression of multiple cytokines in response to proinflammatory mediators and blocked the release of both granulocyte macrophage colony-stimulating factor and granulocyte colony stimulating factor. LXR activation also inhibited proliferation of hASM cells and migration toward platelet-derived growth factor chemoattractant, two important processes that contribute to airway remodeling. Our findings reveal biological roles for LXR in ASM cells and suggest that modulation of LXR activity offers prospects for new therapeutic approaches in the treatment of asthma and other inflammatory respiratory diseases.

HUMAN AIRWAY SMOOTH muscle (hASM) (1) cells are established modulators of the inflammatory process observed in airway diseases such as asthma and chronic obstructive pulmonary disease (COPD) (1, 2). Airway hyperresponsiveness and inflammation characteristic of asthma are associated with increased proliferation of hASM cells and secretion of inflammatory cytokines, growth factors, and various costimulatory molecules, which promote activation, recruitment, and survival of leukocytes, lymphocytes, eosinophils, neutrophils, and monocytes that, in turn, promote airway narrowing and remodeling (3). Current therapeutic regimens, in particular the use of corticosteroids that inhibit expression of proinflammatory factors through the actions of the glucocorticoid receptor, are limited in their effectiveness and can have severe side effects. Identification of novel antiinflammatory agents and therapeutic targets in the airway would thus potentially be of significant clinical value.

Recent findings point to important roles for members of the nuclear hormone superfamily of ligand-activated transcription factors in normal lung biology and in inflammatory processes. Among these are the subfamily of peroxisome proliferator-activated receptors (PPARs) (4). PPARs play fundamental roles in mammalian physiology and disease by controlling the expression of a wide spectrum of genes that serve to govern lipid and metabolic homeostasis, energy utilization, differentiation, proliferation, and inflammation (5). PPAR agonists have been shown to inhibit proinflammatory gene expression and to reduce airway inflammation in animal models of the disease (6, 7).

The liver X receptor (LXR, α and β subtypes) subfamily of nuclear receptors is related to PPARs in biological function and thus, is also potentially relevant in lung biology. LXRs play essential roles in regulating cholesterol homeostasis and lipid metabolism and have been shown to modulate inflammatory gene expression and immune response in macrophages (8–10). LXRs are implicated in the development and etiology of a number of metabolic disorders including diabetes, atherosclerosis, and cardiovascular disease.

LXRs are activated by oxidized derivatives of cholesterol (oxysterols) and function by binding to cognate response elements (LXR-responsive elements; LXREs) of target genes by association with the obligate heterodimerization partner retinoic X receptor α (RXRα) (11). To date, greater than a dozen direct LXR target genes have been identified, most of which are involved in cholesterol and lipid metabolism and reverse cholesterol transport (10). These include members of the ATP-binding cassette (ABC) superfamily of membrane transporters such as ABCA1, ABCG1, ABCG5, and ABCG8, which mediate cholesterol and phospholipid efflux from a variety of cell types, lipogenic enzymes such as fatty acid synthase (FAS) and sterol regulatory element binding protein 1c, lipoprotein remodeling enzymes such as lipoprotein lipase (LPL) and cholesterol ester transport protein, and the glucose transporter (GLUT)4, among others (12–17). LXR is also a key regulator of inflammatory signaling and has been shown to inhibit expression of proinflammatory cytokines including inducible nitric oxide synthase, cyclooxygenase 2 (COX-2), IL-6, monocyte chemotactic protein 1α (MCP-1), and matrix metalloproteinase 9 in macrophages after stimulation with inflammatory mediators (9, 18).

The role of LXR in lung biology is unexplored. Given the established roles of LXR in inflammation, we sought to determine whether LXRs are expressed and functional in primary cultures of hASM cells. We show here that both LXRα and LXRβ are abundantly expressed in hASM cells. LXR agonists stimulated expression of exogenously introduced LXR-responsive reporter genes, as well as endogenous LXR target genes including ABCA1 and ABCG1. In addition, LXR agonists blocked the release of proinflammatory cytokines granulocyte macrophage colony-stimulating factor (GM-CSF), and granulocyte colony stimulating factor (G-CSF), from cells that were challenged with proinflammatory cytokines. LXR activation attenuated cell migration toward platelet-derived growth factor (PDGF) and inhibited proliferation induced by PDGF. These observations establish a role for LXR and hASM cells in modulating expression of proteins involved in the reverse cholesterol transport process, and point to the existence of a novel LXR-modulated antiinflammatory pathway in these cells. LXR may thus represent a potentially new target for therapeutic intervention in inflammatory respiratory diseases such as asthma and COPD.

RESULTS AND DISCUSSION

LXRα and LXRβ Are Functionally Expressed in hASM Cells

To determine whether LXR α and β mRNAs are expressed in hASM cells, total RNA was isolated from primary cells and analyzed by RT-PCR using isoform-specific primers. mRNA for both LXR isoforms was detected in primary hASM cells isolated from three individuals, with LXRβ present at an approximately 5- to 20-fold higher abundance vis-à-vis LXRα (Fig. 1A). The levels of LXR isoforms did not vary significantly among different individuals, and the levels were comparable to those present in a human liver cell line (HepG2) and a human monocytic cell type (THP-1) (Fig. 1A). To determine whether hASM cells produce functional LXR protein, we undertook transient transfection assays of hASM cells using a LXRE-luciferase reporter gene. As shown in Fig. 1B, reporter gene activity was increased 2-fold over control levels in the presence of the synthetic LXR ligand T1317 or the retinoic X receptor (RXR) ligand 9-cis-RA, whereas activity was increased 3- to 4-fold in cells treated with both ligands (results in the figure represent pooled data from independent transfections carried out with hASM cells prepared from three patients). These findings are consistent with the observations that permissive LXR/RXR heterodimers can be activated by ligands for either LXR or RXR and that activity is synergistically enhanced in the presence of ligands for both partners (10, 12). The synthetic agonist T1317 is also reported to activate pregnane X receptor and farnesoid X receptor (19, 20); however, these nuclear receptors are not detectably expressed in hASM cells (Fig. 1C). The specificity of our findings to LXR was further confirmed with transfections carried out with GW3965, a highly selective synthetic LXR agonist (data not shown).

Fig. 1.

LXRα and LXRβ mRNAs Are Functionally Expressed in hASM Cells A, cDNA was prepared from total hASM RNA from three patients or RNA isolated from HepG2 and THP-1 cells, incubated with primers specific for LXRα or LXRβ, and subjected to PCR as indicated. Amplification products were of the expected sizes (173 and 141 bp for LXRα and LXRβ, respectively). Controls included reactions carried out in the absence of template (NTC) or reverse transcriptase (RT). B, LXR ligands activate expression of LXR-responsive reporter genes. hASM cells were transfected with pLXREluc in the presence of T1317 (10 μm) and/or 9-cisRA (10 μm) as indicated, incubated for 48 h, and luciferase activity was measured. The values presented represent the average (±sem) from three independent transfections (each done with cells isolated from different patients) done in triplicate and normalized to protein levels, and to the value obtained from control cells treated with vehicle (taken as 1). *, P < 0.05 as compared with vehicle-treated samples. C, cDNA was prepared as in panel A and subjected to PCR with primers specific for farnesoid X receptor (FXR) or pregnane X receptor (PXR). HepG2 cDNA served as a positive control whereas β-actin levels were used as a loading control.

LXR Agonists Activate Expression of Endogenous LXR-Target Genes in hASM

The foregoing indicates that endogenous LXR is functional as assessed by transient assays. To determine whether LXR agonists modulate expression of bona fide endogenous LXR-target genes, cells were treated with T1317 and/or 9-cis-retinoic acid (9-cisRA), and the expression of known LXR target genes was monitored by real-time PCR. As shown in Fig. 2, A and B, exposure of hASM cells to LXR/RXR agonists stimulated expression of endogenous genes for ABCA1, ABCG1, FAS, LPL, GLUT4, and SR-BI. Induction ranged from 2-fold for SR-BI to approximately 14-fold for ABCA1, ABCG1, and GLUT4. To further confirm that the above findings were mediated by LXR, we examined the expression of representative LXR-target genes ABCA1, ABCG1, and LPL in cells treated with GW3695, a highly specific synthetic LXR agonist. As shown in Fig. 2C, treatment of cells with GW3965 led to the induction of these genes as expected, thereby confirming that the effects were mediated by LXR. Induction of ABCA1 and ABCG1 was also shown at the protein level by Western blot analysis with specific antibodies to human ABCA1 and ABCG1 (Fig. 3A), consistent with the real-time PCR analysis. Although not an exhaustive survey, our findings indicate that LXR activation induces a spectrum of genes in hASM cells that are variously involved in lipogenesis, lipoprotein remodeling, glucose uptake, and cholesterol homeostasis.

Fig. 2.

LXR Activation Induces Expression of Endogenous LXR-Target Genes cDNA was prepared from RNA isolated from three independent preparations of hASM cells incubated in the presence or absence of T1317 and/or 9-cisRA (panels A and C) or GW3965 (2 μm) and/or 9-cisRA, (panel C) where indicated. The resulting cDNA was pooled and subjected to real-time PCR amplification using primers specific to the indicated genes. The values represent the fold-induction above vehicle-treated cells (taken as 1) done in duplicate and normalized using β-actin as an internal standard.

Fig. 3.

LXR Activation Increases ABCA1 and ABCG1 Protein Levels and Stimulates Reverse Cholesterol Transport A, LXR activation induces expression of ABCA1 and ABCG1 proteins. hASM cells were treated for 24 h with T1317 and/or 9-cisRA, and protein extracts were analyzed by Western blot with antibodies specific for human ABCA1, ABCG1, and β-actin as shown. 1, untreated; 2, T1317; 3, 9-cisRA; 4, T1317 + 9-cisRA. B, LXR activation increases cholesterol efflux to HDL and ApoAI. hASM cells were incubated with [³H]cholesterol in the presence or absence of LXR/RXR ligands T1317 (10 μm) and 9-cisRA (10 μm). Cholesterol efflux was then measured in the presence of BSA (0.2%), ApoAI (50 μg/ml), or HDL (50μg/ml) as described in Materials and Methods. C, hASM cells were transfected with siRNA targeting luciferase (negative control) or LXRα and β isoforms as described in Materials and Methods. Cells were then incubated with [³H]cholesterol as in panel B, and cholesterol efflux was measured in the presence of BSA (0.2%) or HDL (50 μg/ml) in supernatants collected after 5 h. N.S., Not significant. D, hASM cells were transfected with siRNA specific to luciferase (negative control) or with a mixture of two siRNAs targeting LXRα and LXRβ isoforms and incubated for 48 h. Cells were treated with T1317 for an additional 24 h, and RNA was isolated and quantified by real-time PCR. The data represent the average of duplicate experiments done in triplicate (±sd) and normalized using β-actin as an internal standard.

The expression and LXR-dependent induction of ABCA1 and ABCG1 are of particular interest. ABCA1 and ABCG1 regulate cholesterol homeostasis by controlling cholesterol and phospholipid efflux from tissues to extracellular lipid-poor acceptors apolipoprotein AI (ApoAI) and high-density lipoprotein (HDL), respectively, for transport to the liver and subsequent catabolism (21, 22). Both ABCA1 and ABCG1 are highly expressed in lung tissue and have been implicated in normal lung physiology, although the roles of these transporters in the lung are poorly understood (23, 24). ABCA1 has been shown to regulate reverse transport of cholesterol and phospholipids to ApoA1 acceptors in alveolar type II cells and to enhance oxysterol-dependent basolateral surfactant efflux from alveolar epithelia (25). ABCA1 null mice die from respiratory failure due to pulmonary edema (26). ABCG1-null mice display profound changes in lung morphology and histology and altered plasma lipid levels and lipid accumulation in subpleural regions (23).

To determine whether hASM cells transport cholesterol in response to LXR activation, hASM cells were incubated with [³H]cholesterol, and efflux to ApoA1 and HDL was measured after stimulation with LXR/RXR ligands. As shown in Fig. 3B, activation of LXR/RXR led to increased reverse cholesterol transport to both HDL and to ApoA1. The ligand-induced efflux was shown to be dependent on the expression of LXRα/β isoforms as determined by short interfering RNA (siRNA)-mediated inhibition of LXR expression (Fig. 3C). Figure 3D confirms that the siRNAs used effectively knocked down expression of the LXR isoforms in hASM cells.

The role of cholesterol in asthma and other inflammatory lung diseases is not fully resolved. There is indirect evidence that cholesterol accumulation may be associated with airway hyperresponsiveness and airway inflammation as seen in asthma. Male C57BL6 mice fed on a diet supplemented with 2% cholesterol showed higher numbers of eosinophils and elevated levels of IL-5, PGE2, and MCP-1 in bronchoalveolar lavage (BAL) fluid after sensitization and inhalation exposure to ovalbumin (27). In addition, dietary cholesterol also resulted in elevated production of IL-4 and IFN-γ by lymphocytes isolated from the lungs. These inflammatory indicators were all significantly correlated with serum cholesterol levels. Cholesterol constitutes approximately 10% of the composition of lung surfactant and is the major neutral lipid of surfactant. Surfactant deficiency can lead to airway closure, which has been postulated to be one of the mechanisms of increased airway hyperresponsiveness. A retrospective analysis demonstrated that serum cholesterol was higher in children with asthma compared with nonasthmatic children (28). However, there is little direct evidence at present that raised serum cholesterol or accumulation of cholesterol in the lung or airways worsens airflow obstruction or airway inflammation.

The most convincing evidence of the role of cholesterol in airway pathophysiology is the accumulation of cholesterol-rich protein in the alveolar spaces in patients with pulmonary alveolar proteinosis. Activity of ABCA1 is important for the maintenance of normal lung lipid composition, structure, and function (26). Although it is thought that cholesterol homeostasis is largely maintained by the alveolar macrophages, our findings that both ABCA1 and ABCG1 are expressed in hASM cells and promote efflux of cholesterol after induction by LXR activation suggests that airway smooth muscle cells may also participate in this process.

LXR Agonists Have Antiinflammatory Effects on hASM Cells

Asthma and other respiratory diseases, such as COPD, are characterized by narrowing of the airways and chronic inflammation (29, 30). ASM cells secrete a variety of cytokines and chemokines in response to inflammatory signals. These mediators include GM-CSF and G-CSF, which promote recruitment and survival of infiltrating eosinophils and neutrophils, leading to further propagation of the inflammatory response and to airway constriction and remodeling.

Because LXR is known to inhibit inflammatory signaling in macrophages and other cell types, we wished to determine whether LXR also displays antiinflammatory properties in hASM cells. hASM cells were incubated in the presence of a mixture of TNFα, interferon-γ (IFNγ), and IL-1β (cytomix), and expression of multiple cytokine/chemokines was determined at the transcriptional level by real-time PCR. As shown in Fig. 4A, treatment of hASM cells with cytomix resulted in the increased expression of MCP-1α, Cox-2, IL-6, RANTES (regulated upon activation normal T-cell expressed and secreted), G-CSF, and GM-CSF as expected. Coincubation of cells with T1317 inhibited cytomix-mediated induction of mRNA expression of all cytokines tested. Inhibition of cytomix-mediated expression by T1317 was similar to that observed with the steroid dexamethasone, an agonist for GR. The only exception was MCP-1α where inhibition T1317 was not statistically significant. The levels of cytomix-induced expression of individual cytokines differed slightly between THP-1 cells and hASM cells as determined by comparison of cycle threshold (Ct) values; however, the level of LXR-mediated repression was similar between both cell types (data not shown). To determine whether reduction of mRNA expression corresponded to reduced protein levels, we examined release of GM-CSF and G-CSF by ELISA. As shown in Fig. 4B, cytomix-stimulated release of G-CSF and GM-CSF was inhibited 30% and 70%, respectively, by coincubation with T1317. To confirm that the above results were dependent upon LXR, the expression of two candidate genes, Cox-2 and IL-6, was analyzed after knockdown of LXR isoforms with transfected siRNA (Fig. 5C). As shown in Fig. 5, A and B, transfection of cells with LXRα/β siRNAs attenuated T1317-mediated repression of Cox-2 and IL-6 after cytomix treatment (Fig. 5, A and B). The foregoing indicates that LXR agonists have antiinflammatory properties in hASM cells.

Fig. 4.

LXR Agonists Inhibit Cytokine Release and Cytokine mRNA Expression in hASM Cells A, hASM cells were serum starved for 48 h and incubated with cytomix (TNFα, IFNγ, and IL-1β) (Ctmx) for an additional 24 h. Where indicated, cells were also cotreated with cytomix and T1317 (10 μm) or dexamethasone (Dex) (10 μm). cDNA prepared from RNA isolated from three independent biological samples for each experimental condition was pooled and subjected to real-time PCR with primers specific for the indicated cytokines. The values represent the fold-induction above vehicle-treated cells (taken as 1) from duplicate experiments and normalized using β-actin as an internal standard. *, P < 0.05 as compared with cytomix-treated samples. B, Supernatants were collected and the levels of extracellular GM-CSF and G-CSF were assessed by ELISA. Values shown represent the percent release relative to cytomix alone (±sd), which was taken as 100%.*, P < 0.05 as compared with cytomix-treated samples. RANTES, Regulated upon activation normal T-cell expressed and secreted.

Fig. 5.

LXRα/β Expression Is Required for the Antiinflammatory Effects of T1317 hASM cells were transfected with siRNA for 48 h with sequences specific to luciferase (negative control; white bars) or with a mixture of two siRNAs targeting LXRα and LXRβ isoforms (black bars). Cells were serum starved for 48 h and incubated with cytomix (TNFα, IFNγ, and IL-1β) (Ctmx) for an additional 24 h. Where indicated, cells were also cotreated with cytomix and T1317 (10 μm). RNA samples were subjected to real-time PCR with primers specific for Cox-2 (panel A) and IL-6 (panel B). C, LXR isoform knockdown was confirmed by real-time PCR. The data represent the average of duplicate experiments done in triplicate (±sd) and normalized using β-actin as an internal standard. N.S., Not significant.

LXR Activation Attenuates hASM Migration and Proliferation

Smooth cell migration is thought to contribute to smooth muscle accumulation in the submucosa and promote airway remodeling in patients with chronic asthma, analogous to vascular smooth muscle migration in atherosclerosis (2). ASM cells migrate toward chemotactic gradients initiated by a variety of cytokines and growth factors. To determine whether LXR agonists modulate migration of hASM cells, we examined PDGF-induced chemotaxis using transwell migration assays. As shown in Fig. 6A, hASM cells showed a 3.5-fold increased chemotaxis toward 1 ng/ml PDGF. Migration was inhibited in a concentration-dependent manner by 70–80% in the presence of T1317. LXR did not decrease the adherence of ASM cells to collagen type I coated wells at concentrations tested in the migration assay, indicating cell viability was not compromised (Fig. 6B). To begin to explore possible mechanisms involved in inhibition of migration, we examined the phosphorylation status of several signaling molecules known to be involved in migration such as Src kinase, Akt, and p38 MAPK (31–33). In this preliminary analysis, we did not observe any modulation of the phosphorylation status of the above signaling molecules when cells were treated with LXR agonists before stimulation with PDGF, indicating that these particular factors may not be involved in the LXR-mediated inhibition of migration that we observed (data not shown). We are currently investigating the role of other signaling pathways.

Fig. 6.

LXR Activation Attenuates Migration and Proliferation of hASM Cells A, hASM cells were incubated in transwell plates in the presence or absence of the indicated concentration of T1317, and migration toward PDGF (1 ng/ml) was measured as described in Materials and Methods. The y-axis shows the number of migrated myocytes (mean from four experiments in duplicate ± sd). B, hASM cells were treated with PDGF (1 ng/ml) alone or in the presence of increasing amounts of T1317, as indicated, and adherence to collagen type I-coated plates was measured as described in Materials and Methods. C, LXR agonists decrease hASM proliferation. hASM cells were treated with PDGF (50 ng/ml) alone or in the presence of increasing amounts of T1317, as indicated, for 48 h and proliferation was assayed by BrdU incorporation as described in Materials and Methods. *, P < 0.05 as compared with PDGF alone.

In addition to migration, hASM cell proliferation is also an important parameter in airway remodeling (34). We therefore examined the role of LXR activation on cell proliferation by measuring bromodeoxyuridine incorporation. As shown in Fig. 6C, LXR agonists decreased proliferation of ASM cells in a dose-dependent manner (Fig. 6C).

In summary, we demonstrate that hASM cells functionally express both LXRα and β isoforms and that activation leads to the regulation of a variety of genes and cellular processes that are important in normal lung cell function and in pulmonary diseases such as asthma. Recent findings showing that arginase II, a gene that has been implicated in asthma, is a direct LXR target, support a role for LXR in pulmonary inflammation (35). Ongoing studies of the mechanisms of action and biological functions of LXR in airway, including studies using animal models of airway inflammation, will provide insights into the importance of LXR in normal airway smooth muscle function and in airway disease.

MATERIALS AND METHODS

Reagents

The synthetic LXR agonist TO901317 (herein after referred to as T1317), the synthetic LXR agonist GW3965, 9-cisRA, IFNγ, TNFα, IL-1β, and human ApoAI were purchased from Sigma-Aldrich (Oakville, Ontario, Canada). PDGF was purchased from Invitrogen Canada (Burlington, Ontario, Canada). Rabbit antibodies to human ABCA1 and ABCG1 were purchased from Novus Biologicals (Littleton, CO). Mouse antibody to human β-actin was purchased from MP Biomedicals (Irvine, CA). [³H]cholesterol was obtained from PerkinElmer (Boston, MA).

hASM Cells

hASM cells were obtained as described previously (36) from human lungs that were resected at St. Joseph’s Healthcare (Hamilton, Canada), after approval from the Institutional Review Board and the consent of patients undergoing resection. Smooth muscle tissue was isolated from disease-free areas of the bronchi. ASM cells were grown in DMEM supplemented with 10% fetal bovine serum, 1% l-glutamine, and 1% penicillin/streptomycin. All experiments were done with cells at passage 5 or earlier.

Analysis of LXR mRNA Expression

Total RNA was isolated from hASM cells using the RNeasy QIAGEN kit (QIAGEN, Chatsworth, CA) according to manufacturer’s instructions. cDNA was prepared from 1 μg of RNA by reverse transcription using a commercially available kit (QIAGEN) according to the manufacturer’s instructions and amplified by PCR with primers specific for human LXRα (forward, 5′-GGAGGTACAACCCTGGGAGT-3′; reverse, 5′-AGCAATGAGCAAGGCAAACT-3′); and LXRβ (forward, 5′-TCACCTACAGCAAGGACGAC-3′; reverse, 5′-AGAAGATGTTGATGGCGATG-3′).

Briefly, reactions contained 12.5 μl of SYBR-green supermix (Invitrogen, Carlsbad, CA), 10.5 μl of H₂O, 1 μl of primer sets (10 μm each), and 1 μl of cDNA. PCR amplification was carried out for 30 cycles with the following parameters: denaturation at 95 C for 15 min; 30 cycles at 95 C for 30 sec and 60 C for 1 min. Products were separated on 2% agarose gels, stained with SYBR green, and imaged on a Typhoon 9200 Variable Mode Imager (Molecular Dynamics, Amersham Biosciences, Baie D’Urfe, Quebec, Canada).

Transfections and Reporter Gene Assay

hASM cells were transfected with 0.4 μg of pLXREluc in six-well dishes at approximately 75% confluency using Effectene reagent (QIAGEN) according to the manufacturer’s instructions. pLXREluc is an LXR-responsive luciferase reporter plasmid that contains three tandem copies of the LXRE from the mouse mammary tumor virus long terminal repeat and has been described elsewhere (37). After transfection, plates were incubated overnight in DMEM lacking phenol red and supplemented with 10% charcoal-stripped fetal bovine serum, 1% l-glutamine, and 1% penicillin/streptomycin with 10 μm T1317 and/or 10 μm 9-cisRA (from stock solutions prepared in dimethyl sulfoxide) for an additional 48 h (fresh ligand was added after 24 h) as described in the figure legends. Control cells received an equivalent amount of vehicle. Luciferase activity was assayed as described previously (38).

Real-Time PCR

hASM cells were incubated in the presence or absence of T1317 and/or 9-cisRA and cytomix (TNFα, 30 ng/ml; IFNγ, 100 ng/ml; IL-1β, 5 ng/ml) as indicated in figure legends. Total RNA was isolated and cDNA was prepared as described above. Real-time PCR was performed using Platinum SYBR Green Supermix-UDG with ROX PCR mix (Invitrogen) according to the manufacturer’s instructions. Briefly, 12.5μl of SYBR-green Supermix, 10.5 μl of H₂O, 1 μl of primer sets (10 μm each forward and reverse primer; specific for human ABC1, ABCG1, LPL, FAS, GLUT4, and SR-BI, respectively) and 1 μl of cDNA was mixed with a final reaction volume of 25 μl. PCR amplification was carried out in 96-well plates in an Applied Biosystems 7900HT real-time PCR machine (Applied Biosystems, Foster City, CA). Relative expression was determined using the standard curve method (39) and by normalizing to β-actin expression levels. Sequences are available upon request.

siRNA Transfection

hASM cells were transfected with 5 nm siRNA oligos against the gene of interest using HiPerfect reagent (QIAGEN) according to manufacturer’s instructions (ratio of 3 μl HiPerfect to 5 nm siRNA). Double-knockdown experiments were performed with a mixture of individual siRNAs. The transfection complex was added to the cells for 48 h, and knockdown and off-target specificity was assessed by real-time PCR. After 48 h, assays were conducted as described for real-time PCR and cholesterol efflux assays. All siRNA oligos were purchased from QIAGEN. LXRα, catalog no. SI00080416; LXRβ, catalog no. SI00094787; luciferase negative control, catalog no. 1022070.

Western Blot Analysis

hASM cells were grown to confluency in 100-mm plates and incubated in the presence of T1317 (10 μm) and/or 9-cisRA (10 μm) for 24 h as indicated in the figure legends. Western blot analysis was carried out with 25 μg total protein for each sample using a commercially available kit (Amersham Pharmacia Biotech, Arlington Heights, IL) according the manufacturer’s instructions. After transfer to nitrocellulose, blots were incubated with rabbit anti-ABCA1 or rabbit anti-ABCG1 polyclonal antibody (1:2000) for 1 h followed by goat antirabbit horseradish peroxidase-conjugated secondary antibody (1:5000) (Amersham) for an additional hour and visualized by enhanced chemiluminescence. Blots were probed with rabbit anti-β-actin as a loading control.

Cholesterol Efflux

Cholesterol efflux was performed as described previously (23). HDL (density between 1.063 and 1.215 g/ml) was purified from human plasma by sequential KBr density gradient centrifugation (40). hASM cells were grown to 90% confluency in six-well dishes and incubated for 48 h in the presence of [³H]cholesterol (5 μCi/ml). Cells were then washed and incubated for an additional 18 h with equilibration medium (DMEM + 2% BSA) supplemented with T1317 (10 μm) and 9-cisRA (10 μm) where indicated. Efflux was initiated by the addition of efflux medium (DMEM) plus either BSA (0.2%), ApoAI (50 μg/ml), or HDL (50 μg/ml) where indicated. Supernatants were collected at various times up to 7.5 h, and the cells were washed and lysed with 0.1 m NaOH for 15 min. Radioactivity in the media and cell extracts was measured by scintillation counting. Cholesterol efflux was calculated as the amount of [³H]cholesterol in the media at the time indicated/total [³H]cholesterol associated with the cells.

ELISA

Confluent hASM cells in 100-mm plates were incubated in DMEM supplemented with 0.3% BSA for 48 h. Cytomix was added with or without T1317 (10 μm) for an additional 24 h. Supernatants were collected, and GM-CSF and G-CSF concentrations were measured using commercially available ELISA kits according to manufacturer’s instructions (R & D Systems, Minneapolis, MN).

Migration Assay

Migration experiments were performed using a 6.5-mm Transwell culture plate with a 8.0-μm pore, collagen-I coated, polycarbonate membrane separating the inner and the outer chambers (Fisher Scientific Ltd., Nepean, Ontario, Canada) as previously described (36). Briefly, confluent smooth muscle cells were maintained in growth factor-free medium for 24 h before the experiments. Cells (100 μl) were treated with varying concentrations of T1317 (1, 10, 50 μm) and plated on the upper side of the membrane, and PDGF (1 ng/ml, 600 μl) was added to the lower wells. After 5 h, the membranes were peeled off and the cells on the upper face of the membranes were scraped using a cotton swab. Cells that migrated to the lower face of the membrane were fixed with 3.7% formaldehyde and stained with Diff-Quik (VWR International, Mississauga, Ontario, Canada). The number of migrated cells on the lower face of the filter was counted in four random fields under ×20 magnification (Olympus BX40 microscope; Sony 3CCD Power HAD video camera, and Northern Eclipse Software from Empix Imaging, Mississauga). Assays were done in duplicate using tissues from four different lung specimens.

Proliferation Assay

hASM cell proliferation was determined using an ELISA-based bromodeoxyuridine (BrdU) incorporation assay according to manufacturer’s instructions (Roche Clinical Laboratories, Indianapolis, IN). Briefly, hASM cells were plated in 96-well dishes at approximately 60% confluency and serum starved for 24 h. Proliferation was induced by the addition of PDGF-BB (50 ng/ml) with varying concentrations of T1317, as indicated in the figure legends, and incubated for 24 h. BrdU (final concentration 10 μm) was then added to each well for an additional 24 h. Cells were then fixed and incubated with an anti-BrdU antibody followed by incubation with substrate solution for 30 min according to manufacturer’s instructions. The colorimetric absorbance readings were performed at 370 nm and corrected for background at 492 nm on a SpectraMax Plus plate reader (Molecular Devices, Sunnyvale, CA).

Adherence Assay

Adherence assays were done as described elsewhere (41). Briefly, 96-well plates were coated with collagen Type I (100 μg/ul solution) and incubated for 2 h at 37 C. Cells (100 μl) at a concentration of 3 × 10⁵ cells/ml were added per well in combination with PDGF alone or increasing concentration of T1317 as indicated in the figure legend. Cells were allowed to adhere for 1 h at 37 C. Cells were then fixed with 4% paraformaldehyde containing 0.5% crystal violet stain for 30 min at 4 C. The plates were washed extensively in PBS to remove excess stain and read at 595 nm on a SpectraMax Plus plate reader.

Statistical Analysis

Unpaired t tests were used for comparison of groups. P < 0.05 was considered significant.

Acknowledgments

This work was supported by grants from the Heart and Stroke Foundation of Ontario (to J.P.C.) and from the Canadian Foundation for Innovation (to K.P.). K.P. holds the Canada Research Chair in Airway Regulation and Inflammation.

Disclosure Statement: C.J.D., P.B., K.R., J.S., B.L.T., and J.P.C. have nothing to disclose. G.C. has consulted for Boerhinger-Ingelheim and Pfizer and has received lecture fees from GlaxoSmithKline and AstraZeneza. K.P. has received lecture fees from GlaxoSmithKline, Merck, and AstraZeneza.

Abbreviations

 
• ABC

  ATP-binding cassette;

 
• ABCA1/G1

  ABC transporter;

 
• ASM

  airway smooth muscle;

 
• ApoAI

  apolipoprotein AI;

 
• BrdU

  bromodeoxyuridine;

 
• 9-cisRA

  9-cis-retinoic acid;

 
• COPD

  chronic obstructive pulmonary disease;

 
• COX-2

  cyclooxygenase 2;

 
• FAS

  fatty acid synthase;

 
• GLUT

  glucose transporter;

 
• GM-CSF

  granulocyte macrophage colony stimulating factor;

 
• G-CSF

  granulocyte colony stimulating factor;

 
• HDL

  high-density lipoprotein;

 
• LPL

  lipoprotein lipase;

 
• LXR

  liver X receptor;

 
• LXRE

  LXR-responsive element;

 
• MCP1α

  monocyte chemotactic protein 1α;

 
• PDGF

  platelet-derived growth factor;

 
• PPAR

  peroxisome proliferator-activated receptor;

 
• RXR

  retinoic X receptor;

 
• siRNA

  short interfering RNA.