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RECEPTORS AND SIGNAL TRANSDUCTION

ROS and NF-B but not LXR mediate IL-1 signaling for the downregulation of ATP-binding cassette transporter A1

Department of Physiology and Pathophysiology, School of Basic Medical Sciences and Key Laboratory of Molecular Cardiovascular Science, Ministry of Education, Peking University, Beijing, People's Republic of China

Submitted 16 January 2006 ; accepted in final form 22 November 2006

	ABSTRACT

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ATP-binding cassette transporter A1 (ABCA1), a pivotal regulator of cholesterol efflux from cells to apolipoproteins, plays an important role in cholesterol homeostasis. As an inflammatory factor, IL-1 has been shown to downregulate ABCA1 in macrophages and facilitates foam cell formation. However, the molecular mechanism underlining the downregulated ABCA1 by IL-1 is still elusive. In the present study, we demonstrated that IL-1 downregulated ABCA1 but not ABCG1 at mRNA and protein levels in a time- and dose-dependent manner in THP-1 and A549 cells. IL-1 attenuated ABCA1 promoter activity through an LXR (liver X receptor)-independent pathway, since IL-1 did not alter the expression and activities of LXR/, and deletion of the LXR responsive element from the ABCA1 promoter failed to reverse the IL-1 effect. In contrast, NF-B inhibition by pyrrolidine dithiocarbamate and MG132 prevented the suppression of ABCA1 by IL-1. Cotransfection with ABCA1 luciferase reporter and the expression plasmids of Rel A decreased ABCA1 promoter activities. An adenovirus expressing NF-B inhibitor subunit- inhibited NF-B activities and also reversed the IL-1 effect at the promoter activity and protein levels of ABCA1. In addition, IL-1 could induce the production of reactive oxygen species (ROS), and N-acetyl-L-cysteine, a scavenger of ROS, reversed the decreased level of ABCA1 induced by IL-1. H₂O₂ decreased ABCA1 at the mRNA and protein levels and the promoter activity. Thus our data provide strong evidence that ROS and NF-B, but not LXR, mediate the IL-1-induced downregulation of ABCA1 via a novel transcriptional mechanism, which might play an important role of proinflammation in the alteration of lipid metabolism.

interleukin-1; nuclear factor-B; reactive oxygen species

It is commonly accepted that the efflux of cholesterol from cells is caused by two different pathways: passive efflux from the cell membrane to HDL and energy-dependent and apolipoprotein-mediated efflux (41). The latter is linked to ATP-binding cassette transporter A1 (ABCA1), a 254-kDa cytoplasmic membrane protein (14). Mutations in the ABCA1 gene, discovered in patients with Tangier disease and familial HDL deficiency, cause impaired efflux of lipids, including free cholesterol and phospholipids to apolipoprotein A-I, which results in a near absence of plasma HDL. Thus ABCA1 plays a key role in maintaining the homeostasis of cellular lipid metabolism by moving out excess intracellular cholesterol and phospholipids. Distribution of ABCA1 mRNA is considered to be wide, with variation in abundance in specific sites, including cells of the small intestine, liver, brain, kidney, and lung (39).

The expression of ABCA1 is highly regulated both at the transcriptional and posttranscriptional levels (26). At the transcriptional level, the direct repeat (DR) 4 element regulates the expression of ABCA1 in response to variations in cellular content of cholesterol and oxysterols. This occurs via factors belonging to the liver X receptor (LXR)/retinoid X receptor (RXR) family (9). Indirectly, peroxisome proliferator-activated receptor- can transcriptionally activate LXR to induce ABCA1 (8). ABCA1 transcription is also regulated by factors independent of LXR/RXR. Membrane-permeable analogs of cAMP stimulate ABCA1 transcription in macrophages by unknown mechanisms (27). Other compounds, such as cytokines, bacterial lipopolysaccharide, verapamil, unsaturated fatty acids, and fluvastatin, have been reported to modulate the expression of ABCA1 mRNA.

Downregulation of ABCA1 is observed following exposure to inflammatory stimuli including IL-1, TNF-, IFN-, and LPS (1, 20, 29, 37, 38), in which the LXR-dependent or -independent pathway and transcriptional/posttranscriptional level regulation were reported to be involved. Given the important role of IL-1 and ABCA1 in inflammation and lipid traffic, the present study demonstrates a novel transcription mechanism through which IL-1 downregulates ABCA1 by ROS-mediated upregulation of NF-B but not LXR in THP-1 cells, a human monocytic leukemia cell line, and A549 cells, a human pulmonary epithelial carcinoma cell line. Both cells play important roles in the inflammatory and immune response.

	MATERIALS AND METHODS

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Chemicals. Recombined human IL-1 was purchased from PeproTech (London, UK). Anti-ABCA1 antibody was purchased from Novus Biologicals (Littleton, CO), and anti-actin antibody was from Abcam (Cambridge, MA). Pyrrolidine dithiocarbamate (PDTC) and MG-132 were purchased from Calbiochem (La Jolla, CA). N-acetyl-L-cysteine (NAC) was purchased from Sigma (St. Louis, MO). Enhanced chemiluminescence detection reagents were from Pierce Biotechnology (Rockford, IL). Cell culture media and supplements were from Hyclone (South Logan, UT). All other chemicals and drugs were purchased from Chinese Chemical (Beijing, China).

Cell culture. THP-1 cells (10801; American Type Culture Collection, Manassas, VA) were grown in RPMI 1640 medium containing 10% FBS, 10 mmol/l HEPES, 1 mmol/l sodium pyruvate, and antibiotics. A549 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% FBS. Cells were kept at 37°C in an atmosphere of 5% CO₂-95% room air. For all experiments, cells were used at 70% to 80% confluence.

Western blot analysis. After treatment, the cells were lysed and total proteins from whole cell lysate and cellular membrane proteins were isolated as described previously (22, 43). An equal amount of proteins was loaded onto 6% bis-acrylamide gels and separated by electrophoresis. Separated proteins underwent immunoblotting to reveal ABCA1 or -actin with the use of respective antibodies.

RT-PCR and real-time quantitative PCR. Total RNA isolation and reverse transcription were performed as described previously (22). The ABCA1 primers were sense, 5'-TTAAACGCCCTCACCAAAGAC-3'; and antisense, 5'-AAAAGCCGCCATACTAAACT-3'. The ABCG1 primers were sense, 5'-ACTGCAGCATCGTGTACTGGA-3'; and antisense, 5'-CGTCTCGTCGATGTCACAGTG-3'. The primers of -actin were sense, 5'-CATCTCTTGCTCGAAGTCCA-3'; and antisense, 5'-ATCATGTTTGAGACCTTCAACA-3'. Real-time quantitative PCR analysis was performed in the model CFD-3240 (CHROMO 4TM, Hercules, CA). Reaction conditions were programmed on a computer linked to the detector for 40 cycles of the amplification step (94°C for 30 s; 58°C for ABCA1/ABCG1, 55°C for -actin both for 30 s, and then 72°C for 30 s). The expression levels were converted to expression rates and calculated as the ratio of cDNA molecules of ABCA1 or ABCG1 to those of -actin.

Transient transfection and luciferase reporter assay. Transfections were performed in 12-well plates with jetPEI reagent in A549 cells according to the manufacturer's instructions (Life Technologies). In the promoter activation study, A549 cells were cotransfected with -galactosidase expression plasmid and the reporter plasmid of the hABCA1 [pABCA1(-928)-luc], its mutation constructs [pABCA1(-928 DR4 mut)-luc], a reporter construct containing three copies of LXR-response element (LXRE x 3 TK-luc), or a reporter construct containing five copies of NF-B binding site (NF-B x 5 TK-luc) with the expression vector of Rel A. After 24 h of transfection, cells were treated with or without IL-1, and the luciferase activity was measured with use of the Luciferase Assay system (Promega, Madison, WI). The results were normalized against -galactosidase.

Measurement of ROS (H₂O₂) generation. The generation of ROS in THP-1 cells was detected with luminol plus horseradish peroxide-derived chemiluminescence in a light-tight box with use of a BPCL Ultra-weak luminescence analyzer (Beijing, China). Photon counts were integrated over 1-s periods and shown on a computer monitor. THP-1 cells were treated with IL-1 for 0 to 90 min in free serum RPMI 1640, then with 10 µg/ml horseradish peroxide and 0.5 mmol/l luminol, and immediately examined. All experiments were repeated five times, and the sum counts reflect the formation of ROS.

Adenoviruses and transfer. The recombinant adenovirus carrying the gene for the NF-B inhibitor subunit- (Ad.IB) and the control adenovirus green fluorescent protein (Ad.GFP) were provided by Dr. F. H. Bach (Harvard Medical School, Boston, MA). Ad.IB was constructed to express the porcine IB gene (ECI-6) (40). The adenoviruses were amplified and titered in 293 cells. The functional titers as determined by plaque assays on 293 cells were 1.58 x 10¹¹ plaque-forming units (pfu)/ml (Ad.IB) and 5.62 x 10¹⁰ pfu/ml (Ad.GFP). For adenoviral infection, confluent cells were exposed to Ad.IB or Ad.GFP at a multiplicity of infection of 200 for 24 h and then were treated with or without IL-1 for 24 h.

Statistics. The results are expressed as means ± SE. Data analysis involved use of GraphPad Prism software. One-way ANOVA, Student-Newman-Keuls test, or unpaired Student's t-test was used as appropriate. P < 0.05 was considered significant.

	RESULTS

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IL-1 downregulated the expression of ABCA1 in THP-1 and A549 cells. To investigate the regulation of ABCA1 by the proinflammatory cytokine IL-1, we examined the protein level of ABCA1 in THP-1 and A549 cells. Cells were incubated with IL-1 (1 to 10 ng/ml) for 24 h, and ABCA1 protein was detected by Western blot analysis. As shown in Fig. 1, A and B, ABCA1 protein expression was reduced by treatment with IL-1 (1–10 ng/ml) in both THP-1 and A549 cells. In addition, RT-PCR analysis revealed that the mRNA level of ABCA1 decreased after doses of IL-1 (1–10 ng/ml) for 6 h in both cell types (Fig. 1, C and D). The downregulation of ABCA1 protein by IL-1 was detected as early as 4 h and remained depressed until 24 h (Fig. 1E). Used as a positive control, the LXR ligand T0901317 (10 µmol/l) increased ABCA1 expression at 24 h.

View larger version (44K): [in this window] [in a new window]   Fig. 1. IL-1 downregulated the expression of ATP-binding cassette transporter A1 (ABCA1) in THP-1 and A549 cells. A: THP-1 cells were incubated with the indicated doses of IL-1 for 24 h; protein levels of ABCA1 were assayed by Western blot analysis. B: A549 cells were incubated with the indicated doses of IL-1 for 24 h; the membrane protein was isolated and assayed for ABCA1 by Western blot analysis. C and D: THP-1 and A549 cells were incubated with the indicated doses of IL-1 for 6 h; mRNA levels of ABCA1 were assayed by RT-PCR analysis. E: THP-1 cells were incubated with IL-1 (10 ng/ml) for the indicated times; protein levels of ABCA1 were assayed by Western blot analysis.

IL-1 inhibited ABCA1 promoter activities.

View larger version (13K): [in this window] [in a new window]   Fig. 2. IL-1 inhibited ABCA1 promoter activities in A549 cells. A549 cells were transfected for 24 h with 0.7 µg hABCA1 promoter luciferase plasmid plus 0.2 µg -galactosidase (-Gal) expression plasmid. A: transfected cells were incubated with IL-1 (10 ng/ml) for the indicated times. B: transfected cells were incubated with the indicated doses of IL-1 for 24 h, and the cell lysates were then collected and measured for the relative luciferase activity adjusted by -Gal activity. Data are means ± SE (n = 3 repeated experiments). *P < 0.05 vs. control.

LXR/ was not involved in the downregulation of ABCA1 by IL-1.

View larger version (30K): [in this window] [in a new window]   Fig. 3. Liver X receptor (LXR)/ was not involved in the downregulation of ABCA1 induced by IL-1. THP-1 cells were incubated with IL-1 (10 ng/ml) for the indicated times; total RNA was isolated and reverse transcribed into cDNA. A: mRNA level of LXR/ was assayed by reverse-transcription PCR. B: A549 cells were transfected for 24 h with 0.7 µg LXR-response element (LXRE)-driven luciferase-reporter vector (LXRE-tk-Luc) plus 0.2 µg -Gal expression plasmid and incubated with the indicated doses of IL-1 for 24 h, and the relative luciferase activity adjusted by -Gal activity was then measured. C: mRNA level of ABCA1 was assayed by real-time PCR. D: mRNA level of ABCG1 was assayed by real-time PCR. E: A549 cells were transfected for 24 h with 0.7 µg pABCA1(-928)-luc or pABCA1-DR4M-luc plus 0.2 µg -Gal expression plasmid and incubated with IL-1 (10 ng/ml) or T0901317 (10 µmol/l) for 24 h, and the relative luciferase activity adjusted by -Gal activity was then measured. Data are means ± SE (n = 3 repeated experiments). *P < 0.05 vs. control.

NF-B was involved in the downregulation of ABCA1 induced by IL-1. NF-B is a central mediator of gene expression induced by proinflammatory and proatherogenic stimuli, including inflammatory cytokines, oxidative stress, and bacterial products. We thus questioned whether the suppressive effect of IL-1 on ABCA1 expression would require activation of NF-B. To examine this possibility, THP-1 cells were incubated with 10 to 30 µmol/l PDTC, a potent NF-B inhibitor, in the presence of IL-1 (10 ng/ml) for 24 h. PDTC treatment significantly prevented the decrease of ABCA1 protein levels induced by IL- (Fig. 4A). MG132, another NF-B inhibitor, can inhibit the ubiquitin-proteasome system, which is involved in the posttranscriptional regulation of ABCA1 (36). We therefore examined the effect of MG132 on the level of ABCA1 mRNA induced by IL-1 through real-time PCR. As shown in Fig. 4B, MG132 (10 µmol/l) prevented the ABCA1 decrease induced by IL-1. As a positive control, IL-1 increased the activity of NF-B-driven luciferase (data not shown).

View larger version (23K): [in this window] [in a new window]   Fig. 4. NF-B was involved in the downregulation of ABCA1 induced by IL-1. A: THP-1 cells were pretreated with or without the indicated concentrations of NF-B inhibitor pyrrolidine dithiocarbamate (PDTC) for 30 min and then incubated with IL-1 (10 ng/ml) for 24 h. The ABCA1 protein levels were analyzed by Western blot analysis. The immunoblots were analyzed by densitometry, and the data were generated as integrated intensity units (n = 3 repeated experiments). B: THP-1 cells were pretreated without or with MG132 (10 µmol/L) for 30 min and then incubated with 10 ng/ml of IL-1 for 6 h. The ABCA1 mRNA level was analyzed by real-time PCR. Data are means ± SE (n = 3 repeated experiments). *P < 0.05 vs. control; #P < 0.05 compared with IL-1 alone.

View larger version (29K): [in this window] [in a new window]   Fig. 5. NF-B was involved in the downregulation of ABCA1 induced by IL-1. A: A549 cells were cotransfected for 24 h with 0.4 µg hABCA1 promoter luciferase plasmid and 0.5 µg Rel A expression plasmid or vector plasmid plus 0.1 µg -Gal expression plasmid. Cells were incubated for 24 h, and the relative luciferase activity adjusted by -Gal activity was then measured. B and C: A549 cells were transfected with 0.7 µg hABCA1 promoter luciferase plasmid or 5x NF-B response element-diverted luciferase plasmid plus 0.2 µg -Gal expression plasmid. Cells were infected with adenovirus inhibitor of B (Ad.IB) or the control virus adenovirus green fluorescent protein (Ad.GFP) at 200 multiplicity of infection (MOI). At 24 h postinfection, cells were treated with or without IL-1 (10 ng/ml) for 24 h, and the relative luciferase activity adjusted by -Gal activity was measured. D: THP-1 cells were infected with Ad.IB or the control virus Ad.GFP at 200 MOI. At 24 h postinfection, cells were treated with or without IL-1 (10 ng/ml) for 24 h. The ABCA1 protein level was analyzed by Western blot analysis. The immunoblots were analyzed by densitometry, and the data were generated as integrated intensity units. Data are means ± SE (n = 3 repeated experiments). eIF5, eukaryotic translation initiation factor 5. *P < 0.05 vs. control (Con); #P < 0.05 compared with IL-1 alone.

ROS mediated the downregulation of ABCA1 induced by IL-1.

View larger version (24K): [in this window] [in a new window]   Fig. 6. Reactive oxygen species (ROS) were involved in the downregulation of ABCA1 induced by IL-1. A: after treatment with IL-1 (10 ng/ml) for the indicated times, ROS production from THP-1 cells was measured by luminol-derived chemiluminescence. The sum counts represent the amount of ROS production. Data are means ± SE (n = 5 repeated experiments). B: THP-1 cells were pretreated with or without the indicated concentrations of ROS scavenger N-acetyl-L-cysteine (NAC) for 30 min and then incubated with IL-1 (10 ng/ml) for 24 h. The ABCA1 protein levels were analyzed by Western blot analysis. The immunoblots were analyzed by densitometry, and the data were generated as integrated intensity units. T0, T0901317. Data are means ± SE (n = 3 repeated experiments). *P < 0.05 vs. control, #P < 0.05 compared with IL-1 alone for 30 min.

View larger version (23K): [in this window] [in a new window]   Fig. 7. The exogenous oxidant H2O2 decreased the expression of ABCA1 in THP-1 cells. A: THP-1 cells were incubated with the indicated doses of H2O2 for 24 h; protein level of ABCA1 was assayed by Western blot analysis. B: A549 cells were transfected for 24 h with 0.7 µg hABCA1 promoter luciferase plasmid plus 0.2 µg -Gal expression plasmid; the transfected cells were incubated with the indicated doses of H2O2 for 24 h, and the cell lysates were then collected and measured for the relative luciferase activity adjusted by -Gal activity. Data are means ± SE (n = 3 repeated experiments). C: THP-1 cells were pretreated without or with the 10 µmol/l T0901317 for 30 min and then incubated with IL-1 (10 ng/ml) for 24 h. ABCA1 mRMA level was analyzed by real-time PCR. Data are means ± SE (n = 3 repeated experiments). *P < 0.05 vs. control.

	DISCUSSION

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We have come to appreciate a prominent cross talk between inflammation and lipid metabolism, in which inflammatory mediators alter the expression of key molecules regulating lipid metabolism (7, 18). Recruited in the arterial intima, monocytes acquire the morphological characteristics of macrophages and then accumulate cholesteryl esters in cytoplasmic droplets to lead ultimately to foam cell formation. To maintain the homeostasis of lipid macrophages, ABCA1 promotes the transport of cholesterol and phospholipids to apolipoprotein A-I (33). Endotoxin and cytokines have been reported to decrease ABCA1 expression and cholesterol efflux from cholesterol-loaded macrophages (5, 20, 37). In the inflammatory condition, the breakdown of the homeostatic mechanism aggravates the lesions of artherosclerosis. Especially, IL-1 is a cytokine involved in infection and inflammation (10). Because of its proinflammatory effects on endothelial cells, smooth muscle cells, and macrophages and its production by all of these cell types in atherosclerotic lesions, IL-1 is one of the first cytokines to be considered instrumental in the propagation of vessel wall inflammation in atherosclerosis (35). Thus the downregulation of ABCA1 may be one of the mechanisms by which IL-1 contributes to atherosclerosis. Besides being produced by macrophages, ABCA1 is in metabolically active tissues, such as liver, intestine, and kidney, and also participates in the regulation of the plasma lipid level. Thus the alteration of ABCA1 expression by the inflammatory pathway can have vast implications.

One important intracellular mediator of the IL-1 response is NF-B (3). NF-B is also a critical regulator involved in the cross talk between the inflammatory pathway and nuclear receptor signaling (18). We investigated whether NF-B was involved in the downregulation of ABCA1 induced by IL-1. First, a potent inhibitor of NF-B activation, PDTC, prevented the decrease of IL-1-induced ABCA1 protein level. PDTC is an activator of activator protein-1 (AP-1) as well (21), but no transcriptional effect has been found with AP-1 motifs on the ABCA1 promoter (28, 42). MG132 can inhibit proteasome, which is involved in the posttranscriptional regulation of ABCA1 (36). Therefore, we examined the effect of MG132 on the mRNA level of ABCA1 induced by IL-1 through real-time PCR analysis. MG132 prevented the IL-1-induced decrease of ABCA1 at the mRNA level. Cotransfection with ABCA1 luciferase reporter and the plasmid of Rel A further demonstrated that NF-B plays an essential role in the downregulation of ABCA1 induced by IL-1. Overexpression of IB by adenovirus confirmed the role of NF-B in the regulation of ABCA1. When NF-B was blocked, the basal level of ABCA1 was increased, which indicates that NF-B has a regular effect on the maintenance of ABCA1 basal expression. IL-1 still had a minor, although not significant, tendency to reduce ABCA1 level when NF-B was blocked, which suggests that another mechanism may exist. Baranova et al. (5) reported, too, that the mechanism of the LPS inhibitory effect on scavenger receptor B1 and ABCA1 depends on NF-B in RAW 264.7 cells. However, whether NF-B can directly interact with the promoter binding sequences blocking ABCA1 gene transcription or operate through some intermediate mechanism that can downregulate gene transcription remains to be determined. A coregulator may be one of the candidates. As we know, the coactivators steriod receptor coactivator-1 and p300 and corepressors silent mediator of retinoic acid receptor and thyroid receptor (SMRT) and nuclear receptor compressor have been shown to influence LXR-dependent ABCA1 transcription (15, 17). Perhaps NF-B might influence some coregulator that regulates LXR-independent ABCA1 transcription. cAMP response element-binding protein binding protein and p300 not only are the coactivators of NF-B but also possess intrinsic histone acetyl transferase activity (4, 25). Acetylation of histones is associated with relaxed chromatin configuration, which refers to chromatin remodeling and is thought to facilitate transcription factor access to DNA. Recently, chromatin remodeling was found to be involved in the activation of ABCA1 transcription (16). Thus we presume that IL-1 may suppress ABCA1 in part by altering the endogenous NF-B activity and that NF-B may regulate ABCA1 indirectly through regulating the interaction patterns of some transcription factors or coactivators.

ROS (such as superoxide and H₂O₂) can function as second messengers at low levels and activate signaling pathways resulting in a broad array of physiological responses, also referred to as nonlethal or sublethal oxidative stress. As demonstrated in the present study, IL-1 induces the production of ROS in THP-1 cells within 30 min before ABCA1 mRNA level is decreased. In addition, NAC, a scavenger of ROS, reverses in part the decreased ABCA1 protein level induced by IL-1. The exogenous oxidant H₂O₂ also decreases ABCA1 expression at both the mRNA and protein levels and the activity of ABCA1 promoter. These data suggest that increased oxidative stress may be involved in the downregulation of ABCA1 induced by IL-1. Specific targets affected by ROS are redox-sensitive transcription factors. NF-B is redox sensitive, and its activity is due to the production of ROS by all agonists (32), which initiates a cascade finally leading to a translocation of the p50/p65 subunits into the nucleus (32). Therefore, downregulation of ABCA1 by ROS might be through an NF-B pathway. As we know, the posttranscriptional regulation of ABCA1 is as important as transcriptional regulation. Phosphorylation of ABCA1 has been reported to be involved in maintaining ABCA1 stabilization (23). Besides being an activator of NF-B, ROS might be associated with the stability of ABCA1 by oxidative modification.

The regulation of ABCA1 is complex (26). To date, no universal mechanism explains the downregulation of ABCA1 by inflammatory mediators. In mouse kidney and in HK-2 cells, LPS, IL-1, and TNF- suppress LXR and RXR expression (38). The decrease in LXR/RXR is associated with a decrease in the expression of several LXR target genes involved in cholesterol and fatty acid metabolism (apolipoprotein E, ABCA1, ABCG1, and sterol regulatory element-binding protein-1c). In mesangial cells, IL-1 promotes intracellular lipid accumulation by inhibiting cholesterol efflux through the PPAR-LXR-ABCA1 pathway (29). In rodent liver, LPS decreased mRNA levels of ABCG5 and ABCG8 by downregulating LXR (20). However, in macrophages, although the precise mechanism is unclear, some other work has shown that LXR is not involved in the suppression of ABCA1 by inflammatory mediators (5, 20, 37). In the present study, we report that downregulation of ABCA1 by IL-1 is mediated through an LXR-independent mechanism. First, in THP-1 cells, treatment of IL-1 did not decrease the mRNA expression of LXR and LXR, and IL-1 treatment did not alter the activity of the LXRE-driven luciferase reporter. Second, the level of ABCA1 but not ABCG1 mRNA was decreased with IL-1 treatment. If LXR is involved, the regulatory pattern of ABCA1 and ABCG1 should be similar (24). Finally, the deletion of the DR4 element from the promoter of ABCA1 did not reverse the effect of IL-1. Perhaps the different regulatory pattern depends on the cell type. Kidney and liver belong to metabolically active tissues, and the metabolic nuclear receptors are more active. In inflammatory cells, such as monocytes, nuclear factors involved in inflammation are more sensitive to an inflammatory mediator. However, although both LPS and proinflammatory cytokines can downregulate ABCA1 in monocytes/macrophages, they perhaps share a distinct mechanism. For example, Castrillo et al. (7) demonstrated that lipid A and polyinosinic-polytidylic acid (microbial activators of Toll-like receptor-3 and -4 signaling pathways) inhibit LXR-dependent gene expression, but this cross talk is likely not shared with other inflammatory signal pathways such as IL-1 and TNF-.

As mentioned, most studies suggest that proinflammatory factors, including IL-1, TNF-, IFN-, and LPS (1, 20, 29, 37, 38), inhibit lipid efflux by decreasing the expression of ABCA1, and anti-inflammatory factors, such as IL-10 and aspirin (30, 34), increase ABCA1 expression. Paradoxically, a recent study by Gerbod-Giannone et al. (11) has shown that TNF- acting via NF-B induces ABCA1 expression in macrophages, which represents a beneficial response of phagocytes in the context of atherosclerosis. The discrepancy between the authors' work and our findings may result from different cell types and stimuli used. ABCA1 levels increase in monocytes differentiated to macrophages (2). Our work involved undifferentiated monocytic THP-1 cells. As we know, in the initial process of atherosclerosis, most cells infiltrating in the vessel wall are monocytes. Therefore, we mainly focused on how ABCA1 is regulated by IL-1 in the initial course of atherosclerosis. However, Gerbod-Giannone et al. (11) used differentiated macrophages, which are abundant in advanced plaque, where apoptotic macrophages and released cholesterol may be taken up by other phagocytic macrophages. The potential pathophysiological relevance of the authors' observations may be that a compensatory mechanism is helping to limit plaque development. As well, Gerbod-Giannone et al. (11) have yet to find an exact NF-B binding site. The different roles of NF-B from their findings and ours further indicate that the role of NF-B may be indirectly and nonspecifically mediated. Under different stimuli and environments, NF-B activation may affect other transcriptional factors or coregulators for a different pattern of signaling conduction. Thus further work is needed for a better understanding of the mechanism of NF-B in the regulation of ABCA1.

In summary, we demonstrate that proinflammatory cytokine IL-1 decreases ABCA1 expression by the ROS-NF-B pathway, which is a common pathway of IL-1 contributing to inflammation. These observations provide new insight into the cross talk between inflammatory signaling pathways and lipid metabolism. In consideration of the crucial role of ABCA1 in regulation of HDL metabolism and cholesterol homoestasis, our study contributes to knowledge of a novel mechanism through which IL-1, a proatherogenic factor, might lead to increased foam cell formation and accelerated atherosclerosis.

	GRANTS

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This study was supported by the Major National Basic Research Program of the People's Republic of China (No. 2006CB503802) and the National Natural Science Foundation of People's Republic of China (No. 30330250) and Program for Changjiang Scholars and Innovative Research Team in University (awarded to X. Wang).

	FOOTNOTES

 

Address for reprint requests and other correspondence: X. Wang or Y. Zhu, Dept. of Physiology and Pathophysiology, School of Basic Medical Sciences and Key Laboratory of Molecular Cardiovascular Science, Ministry of Education; Peking Univ., Beijing 100083, People's Republic of China [e-mail: xwang{at}bjmu.edu.cn (for X. Wang) or zhuyi{at}bjmu.edu.cn (for Y. Zhu)]

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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