Identification of a γ-Interferon–Responsive Element in the Promoter of the Human Macrophage Scavenger Receptor A Gene
Abstract—In the present study, we demonstrate γ-interferon (γ-IFN)–inducible scavenger receptor A (SR-A) mRNA expression during the early stages of THP-1 and blood monocyte differentiation. Predominant induction of SR-A type II mRNA parallels the increased accumulation of cholesteryl esters under these conditions. A potential signal transducer and activator of transcription (STAT1) binding site (γ-interferon activation site) in the SR-A promoter demonstrates γ-IFN–inducible DNA binding activity and is most likely responsible for the γ-IFN–dependent expression of an SR-A promoter–luciferase fusion construct. In contrast, γ-IFN inhibits SR-A expression in mature macrophages as well as after prolonged γ-IFN incubation of THP-1 monocytes. Taken together, these results demonstrate opposite effects of γ-IFN on SR-A expression and activity during the early versus late stages of monocyte maturation. γ-IFN–induced STAT1 activation, leading to increased SR-A expression, could therefore play an important role in the initial steps of foam cell formation and xanthomatosis.
- Received January 10, 2001.
- Accepted February 16, 2001.
Monocyte-derived macrophages are implicated in the development of foam cells of atherosclerotic lesions.1 The human macrophage scavenger receptor A (SR-A) binds and internalizes modified LDL, which ultimately results in the accumulation of cholesterol esters in macrophages.2 These results, together with the elevated plasma cholesterol levels and the reduced size of atherosclerotic lesions seen in SR-A/apolipoprotein E (apoE) double-knockout mice,3 have led to the hypothesis that SR-A plays an important role in the formation of foam cells during the early stages of atherosclerosis.
In humans, SR-A expression is almost entirely restricted to macrophages, and it increases during monocyte maturation.4 The 2 SR-A mRNA isoforms result from alternative splicing of the same transcript from a single gene,5 but the functional significance of the 2 isoforms remains unknown.6 Previous studies have demonstrated that during early monocyte differentiation, SR-A expression is stimulated by macrophage colony-stimulating factor.7 This transcriptional activation of the SR-A gene is thought to be mediated by members of the activator protein (AP)-1 and ets family of transcription factors.8 9 In contrast, γ-interferon (γ-IFN), transforming growth factor-β1, and tumor necrosis factor-α have been shown to inhibit SR-A expression in mature macrophages.10 11 12 13 Recent findings have demonstrated that γ-IFN can inhibit transcription of the macrophage SR-A gene by antagonizing the AP-1 and ets transcription factors as a result of competition between AP-1/ets and a signal transducer and activator of transcription (STAT1) for limiting amounts of the transcriptional cofactors CREB-binding protein and p300.11
We have recently reported that SR-A–overexpressing monocytes from a normolipidemic patient with xanthomatosis demonstrated elevated STAT1α and γ-IFN–inducible protein-10 (IP-10) expression.14 Similar results were obtained from a number of subjects with familial hypercholesterolemia, who frequently develop xanthomatosis.14 We now report that γ-IFN regulates SR-A expression in a differentiation-dependent manner in THP-1 and blood-derived monocytes. In agreement with previous reports, γ-IFN exhibits an inhibitory effect on SR-A mRNA expression in mature macrophages.10 11 In contrast, γ-IFN stimulates SR-A expression at early stages of monocyte differentiation. Gel retardation and transfection experiments suggest that γ-IFN–inducible STAT1 binding to a potential STAT1 element (γ-interferon activation site [GAS]) at position −133 to −125 (SR-Ap) in the SR-A promoter is in part responsible for the induction of SR-A expression and activity in γ-IFN–activated monocytes. Our results explain previously discordant findings on the role of γ-IFN in SR-A regulation and provide the first demonstration that a GAS in the SR-A promoter has a regulatory function on SR-A activity.
Human THP-1 monocytes were grown in RPMI 1640 medium containing 10% heat-inactivated fetal calf serum. To promote differentiation, monocytes were incubated with phorbol 12-myristate-13-acetate (PMA 0.1 μg/mL, Sigma). Human recombinant γ-IFN (Gibco-BRL) was added to PMA-induced THP-1 cells at a concentration of 25 ng/mL of medium (175 U/mL). Blood-derived monocytes were isolated from healthy normolipidemic laboratory personnel (ages 30 to 59; 7 men and 5 women) as described.15 This research was approved by the Institutional Ethics Committee, and informed consent was obtained from all subjects.
RNA Extraction and Northern Blot Analysis
Total RNA was isolated from 5 to 8×106 THP-1 cells with Trizol reagent (Gibco-BRL) and analyzed by Northern blotting as described.14 Hybridization was performed with random-primed 2000-bp HindIII-XbaI human SR-A type II (nucleotides −46 to +198216 ) or 490-bp glyceraldehyde 3-phosphate dehydrogenase (nucleotides 3724 to 422414 ) DNA probes.
Total RNA was reverse-transcribed (RT) with murine Moloney leukemia virus reverse transcriptase (Gibco-BRL) as described.14 15 Simultaneous amplification of a 290-bp SR-A cDNA fragment and a 376-bp glyceraldehyde 3-phosphate dehydrogenase cDNA fragment was performed in duplicate by using primers labeled with Hex fluorescence (University of Calgary Core DNA Services, Calgary, Alberta, Canada) as described.15 Determination of STAT1α and IP-10 transcript levels was performed by semiquantitative polymerase chain reaction (PCR) analysis and has been described in detail14 (also see http://www.atvb.ahajournals.org).
Gel Mobility Shift Assays
Preparation of nuclear extracts has been previously described in detail.14 Oligonucleotides comprising potential GAS elements from −133 to −125 (SR-Ap: 5′-TTAGATTTTGCAAAACGTC-3′), −813 to −805 (SR-Ad: 5′-CTCCTGGGTTCAAGCAATTC-3′), and the AP-1 binding site from −67 to −50 (5′-AATGTGTCATTTCCTTTC-3′) of the human SR-A gene8 9 were used to identify the binding of transcription factors. GAS elements from the human GTP-binding protein and IP-10 promoter regions14 served as STAT1 binding controls. One to 2 microliters of nuclear extract (4 μg protein) and [γ-32P]dATP-labeled (Amersham, 800 Ci/mmol) double-stranded oligonucleotides (0.5 ng, 2×105 counts per minute) were incubated for 30 minutes, and protein-DNA complexes were electrophoresed on 6% polyacrylamide gels. A 100-fold excess of unlabeled double-stranded human GTP-binding protein and IP-10 GAS elements or a synthetic AP-1 oligonucleotide (5′-GATCCGATGAGTCAGCCA-3′)14 was added in some experiments.
Transfection of THP-1 Cells
Human SR-A promoter fragments from positions −290 to +54 (344 bp, pSRA-290) and from −116 to +54 (170 bp, pSRA-116) were cloned into pGL3-Basic (Promega). Cells (4×107) were cotransfected with 32 μg of pSRA-290 or pSRA-116 together with 1 μg of β-galactosidase reporter plasmid as an internal control by using the diethylaminoethyl-dextran method (Stratagene). Forty-eight hours after transfection, 107 cells/mL were plated and incubated for 24 to 72 hours at 37°C under various conditions. Luciferase activity was assayed with the Promega luciferase assay system with a Berthold LB 953 luminometer and normalized to the β-galactosidase standard.
Lipid Loading of Cells
Preparation and acetylation (Ac) of LDL were performed and confirmed by agarose gel electrophoresis as described.14 15 Ac-LDL (50 μg/mL) was added to PMA-activated THP-1 cells (with or without γ-IFN) in RPMI 1640 containing 10% lipoprotein-deficient serum. Cells were incubated for 24 hours at 37°C, washed with phosphate-buffered saline, and collected for lipid extraction.17 Lipids were analyzed by capillary gas chromatography18 (for details of lipid analysis, see http://www.atvb.ahajournals.org).
Accumulation of 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyane (DiI) Ac-LDL (L-3484, Molecular Probes) was analyzed by fluorescence-activated cell sorting flow cytometry (FACScan, Becton-Dickinson) at 555 nm as described.15 PMA-activated THP-1 cells (106) were incubated in 10% lipoprotein-deficient serum with or without γ-IFN for 24 hours, followed by incubation with 10 μg/mL DiI–Ac-LDL for 1 hour at 37°C. Cells were washed 3 times with ice-cold phosphate-buffered saline and subjected to FACScan analysis.15
Ac-LDL was radiolabeled with 125I by the iodine monochloride method.19 THP-1 cells (2 to 3×105) were preincubated with lipoprotein-deficient medium for 30 minutes at 37°C, and 125I–Ac-LDL was added at 4 to 5 μg/mL (in triplicate) for 24 to 72 hours at 37°C. A 50-fold excess of unlabeled Ac-LDL was added to every third sample to determine nonspecific uptake and degradation of 125I–Ac-LDL.20 The remaining cells were lysed with 0.1N NaOH, and total cell protein content was determined.17
γ-IFN Induces SR-A Expression During Early Differentiation of Monocytes
SR-A mRNA levels were determined by RT-PCR with the use of primers corresponding to exons 1 to 3, which are common to both isoforms of SR-A.15 As reported previously,9 13 SR-A mRNA expression was markedly enhanced in PMA-differentiated THP-1 monocytes compared with untreated controls (Figures 1A⇓ and 2A⇓). Addition of γ-IFN resulted in a significant increase (P<0.01) of SR-A expression compared with PMA-activated cells (Figures 1A⇓ and 2B⇓) and was most pronounced (4-fold) at earlier time points (6 to 12 hours). When THP-1 monocytes were preincubated with PMA for various times (6 to 144 hours) followed by γ-IFN incubation for an additional 24 hours, SR-A expression increased only 2.0- to 2.5-fold during the early stages of PMA activation (P<0.01 at 6 hours) and decreased after prolonged incubation with PMA (Figure 1B⇓).
Differential Stimulation of SR-A Isoforms in γ-IFN–Activated Monocytes
γ-IFN–inducible expression of SR-A during early monocyte differentiation was confirmed by Northern blotting (Figure 2⇑). During the first 48 hours of PMA activation, a constant increase of the dominantly expressed SR-AII, as well as the SR-AI isoform, was observed (Figure 2A⇑, lanes 1 through 3). In contrast, PMA incubation for 4 to 7 days reduced SR-AI and SR-AII expression levels 1.5- and 2-fold, respectively, compared with 24-hour PMA-activated THP-1 cells (Figure 2A⇑; compare lane 3 with lanes 4 and 5). Addition of γ-IFN after 6 hours of PMA preincubation resulted in a 2-fold increase of SR-AII mRNA levels after 24 hours (Figure 2B⇑, lane 3), whereas prolonged γ-IFN exposure resulted in a strong decrease in SR-A mRNA levels (Figure 2B⇑, lane 4; also see Figure 2C⇑, lane 5).
γ-IFN–induced SR-A mRNA expression peaked after 12-hour preincubation with PMA (Figure 2C⇑, lane 3), similar to the results obtained from RT-PCR analysis (see Figure 1B⇑) and indicating that maximum γ-IFN–mediated SR-A induction occurs within the first 12 hours of PMA activation. In contrast, prolonged PMA prestimulation resulted in a γ-IFN–mediated repression of both SR-AI and SR-AII mRNA levels (Figure 2C⇑, lanes 4 and 5). Because the duration of PMA incubation mimics the differentiation status of THP-1 cells, our results suggest that the time-related effects on total SR-A expression reflect the sum of differential γ-IFN responses on each SR-A mRNA isoform in early and later stages of monocyte maturation.
γ-IFN Induces SR-A Expression During Maturation of Blood-Derived Monocytes
Human blood–derived monocytes (n=12) were analyzed to confirm the potential relevance of γ-IFN signaling on SR-A expression during blood monocyte maturation (Figure 3⇓). Owing to the variation in SR-A expression among monocytes from different subjects,15 SR-A mRNA increased by 8.5- to 22-fold during maturation of macrophages, depending on the individual analyzed. In contrast and similar to previous findings,10 prolonged incubations with γ-IFN of more mature macrophages resulted in SR-A mRNA expression levels that were reduced by 43% to 80% compared with the respective control macrophages analyzed (P=0.01). These results are correlated with the data obtained from PMA-activated THP-1 monocytes (see Figure 1⇑) and indicate that γ-IFN exerts opposite effects on SR-A expression during early versus late stages of blood monocyte maturation.
Induction of SR-A Activity in γ-IFN–Activated THP-1 Monocytes
To determine that upregulation of SR-A mRNA corresponded to increased SR-A receptor activity, we measured lipid accumulation and Ac-LDL degradation in γ-IFN–activated and PMA-treated THP-1 monocytes (Table 1⇓). γ-IFN activation of PMA-differentiated THP-1 monocytes showed an almost 2-fold increase in cholesteryl ester content after lipid loading (Table 1A⇓; 88.6±10.4 nmol cholesteryl ester–derived cholesterol per mg cell protein; P=0.03), a 2-fold increase in DiI–Ac-LDL accumulation (Table 1B⇓), and a 1.5-fold increase in 125I–Ac-LDL degradation (Table 1C⇓; 154.9±26.7 ng/mg cell protein; P=0.02) compared with PMA-induced controls. These results indicate that γ-IFN–induced SR-A mRNA expression leads to elevated SR-A activity during the early stages of monocyte differentiation.
Expression of Other γ-IFN–Inducible Genes in THP-1 Monocytes
To compare the γ-IFN–regulated SR-A expression pattern with other well-known γ-IFN–responsive genes, we examined the expression of STAT1α and IP-10, a STAT1-responsive gene14 (see Figure⇑ I at http://www.ahajournals.org). In these experiments, the γ-IFN–inducible expression patterns of STAT1α and IP-10 in THP-1 and blood-derived monocytes were similar to the SR-A expression analysis described above and indicated that comparable transcriptional mechanisms are involved in the regulation of these genes.
Identification of a Potential STAT1 Binding Element in the SR-A Promoter
The observed expression pattern of SR-A (Figures 1 through 3⇑⇑⇑) and the increased STAT1α binding activity in γ-IFN–activated THP-1 monocytes14 suggested the presence of potential STAT1 binding sites (GAS) in the 5′ regulatory region of the human SR-A gene. We identified 2 putative GASs, located −125 to −133 (SR-Ap) and −805 to −813 (SR-Ad) upstream from the transcriptional SR-A initiation site, and performed gel retardation assays with nuclear extracts from THP-1 cells and human blood–derived monocytes (Figure 4⇓). The SR-Ad oligonucleotide did not demonstrate γ-IFN–inducible binding of proteins (data not shown). In contrast, extracts from PMA-activated THP-1 monocytes showed γ-IFN–inducible binding activity to the STAT1 recognition site SR-Ap (Figure 4A⇓; compare lanes 4 and 5). This γ-IFN–inducible binding activity was competed efficiently at all time points analyzed by the human GTP-binding protein and IP-10 STAT114 binding elements (Figure 4A⇓, lanes 1 and 2 at t=9 hours). Binding activity peaked after 12 hours (Figure 4A⇓, lane 6), whereas prolonged incubation with γ-IFN resulted in reduced binding (Figure 4A⇓, lanes 7 and 8). Nuclear extracts from blood-derived monocytes also demonstrated γ-IFN–inducible binding at the early stages of maturation only (Figure 4B⇓). These results indicate that γ-IFN–inducible STAT1 binding to the SR-Ap GAS in the SR-A promoter can occur during both THP-1 cell and blood monocyte differentiation.
Differential Regulation of the SR-A Promoter in γ-IFN–Activated Monocytes
THP-1 cells were transfected with a human SR-A promoter–luciferase fusion construct including both the putative GAS and AP-1 sites (pSRA-290) or a deletion mutant containing the AP-1 site only (pSRA-116) (Figure 5⇓). Overnight incubation of transfected PMA-activated THP-1 monocytes with γ-IFN resulted in a 3.1-fold induction of transcriptional activity of the 290-bp SR-A promoter compared with PMA-activated controls (Figure 5⇓; P<0.01). Deletion of the putative SR-Ap GAS in the SR-A promoter construct pSRA-116 led to a complete loss of γ-IFN response after PMA activation. In contrast, but in agreement with other findings,10 11 transfection of the 290-bp SR-A promoter–luciferase fusion construct followed by prolonged (3-day) incubation with γ-IFN resulted in a 3.9-fold reduction of luciferase expression compared with PMA controls (data not shown). Taken together, these data strongly indicate that γ-IFN–induced STAT1 binding to the proximal SR-A promoter region is in part responsible for the increased SR-A expression in PMA-differentiated THP-1 monocytes.
A number of cytokines have been shown to regulate SR-A expression and activity in vitro.8 9 10 11 12 13 The presence of γ-IFN–secreting T lymphocytes in atherosclerotic lesions21 and the important role of γ-IFN for monocyte activation have resulted in several studies that have analyzed the effect of γ-IFN on SR-A expression in macrophages. In those experiments, prolonged incubation of PMA-differentiated THP-1 macrophages or mature primary macrophages with γ-IFN resulted in the downregulation of SR-A expression, followed by reduced Ac-LDL binding and internalization.10 11
In this study, we have demonstrated that γ-IFN can exert opposite effects on SR-A expression, depending on the differentiation status of monocytes and the duration of γ-IFN activation. SR-A mRNA expression was increased after a 24-hour γ-IFN activation of THP-1 monocytes preincubated with PMA for 6 to 12 hours. Results from γ-IFN–activated (<48 hours) human blood–derived monocytes confirm these observations and can be correlated with the increased Ac-LDL degradation of human macrophages after a 22-hour incubation with γ-IFN.22 The dominantly expressed SR-AII mRNA isoform seems responsible for the γ-IFN–induced SR-A activity in γ-IFN– and PMA-activated THP-1 monocytes compared with controls.
Similar to previous findings,10 11 γ-IFN was discovered to inhibit SR-A expression in mature blood-derived macrophages but does not stimulate SR-A mRNA levels in THP-1 cells after prolonged PMA activation. The complex response in SR-A expression after short- versus long-term exposure to γ-IFN cannot be explained by a transcriptional mechanism only. γ-IFN–mediated stimulation of SR-A, affecting predominantly the SR-AII isoform, could reflect functional specificities of the 2 isoforms in acute and chronic exposure to γ-IFN. Furthermore, the diminution of SR-A expression after chronic γ-IFN exposure could be attributable in part to an mRNA-destabilizing effect similar to the tumor necrosis factor-α–mediated posttranscriptional inhibition of SR-A expression in THP-1 cells.13
The potential STAT1 binding site, located at positions −125 to −133 of the SR-A promoter, demonstrated γ-IFN–inducible binding of nuclear proteins. This binding is correlated with γ-IFN–inducible expression of the endogenous SR-A gene and activation of the SR-A promoter reporter gene construct. These findings coincide with maximal induction of the γ-IFN receptor in PMA-activated THP-1 cells under these conditions,23 demonstrating an enhanced capacity of monocytes to respond to γ-IFN. Our results strongly suggest that STAT1 binding to the SR-A promoter is partly responsible for γ-IFN–mediated activation of the SR-A promoter. Although transcriptional activation by AP-1 is thought to be inhibited by STAT1, we did not observe reduced binding of AP-1 to the SR-A AP-1/ets motif after γ-IFN activation during early monocyte maturation (data not shown). These findings suggest that the basal AP-1 activity-dependent transcriptional machinery is not negatively affected by STAT1 activation after a short γ-IFN incubation. The γ-IFN– and PMA-inducible expression of an SR-A promoter–luciferase fusion construct confirms these observations and indicates that adjacent AP-1/ets and STAT1 binding sites on the SR-A promoter may cooperate to induce SR-A expression in early differentiating monocytes. To the contrary, and as shown previously,11 prolonged γ-IFN exposure leads to inhibition of AP-1/ets transcription factors, which results in reduced SR-A promoter activity in mature macrophages.
The opposite effects of γ-IFN on SR-A expression during early versus late stages of monocyte maturation could explain some of the conflicting results obtained from studies on the potential role of γ-IFN in the pathogenesis of atherosclerosis. Decreased SR-A expression in mature macrophages10 11 and inhibition of smooth muscle cell proliferation24 could reflect the antiatherogenic properties of γ-IFN. Conversely, γ-IFN potentiates atherosclerosis in apoE-knockout mice25 and stimulates vascular cell adhesion molecule-1 expression in rabbit aortic endothelium.26 A critical step in the initiation of atherosclerosis is the recruitment of monocytes to the arterial wall.27 Therefore, the stimulatory effect of γ-IFN on SR-A expression could indicate that SR-A might not function as a lipoprotein receptor during the early stages of monocyte differentiation but rather as an adhesion molecule, as proposed by Fraser and coworkers.28 γ-IFN–inducible STAT1 activity in the promoter of the intercellular adhesion molecule-1 gene stimulates adhesion of monocytes29 and suggests a similar role of STAT1 for SR-A regulation and function. Future experiments should be carried out to investigate SR-A’s function as part of a general STAT1-mediated transcriptional mechanism to induce adhesion of monocytes and promote their differentiation to macrophages.
This work was funded by the Medical Research Council/Novartis Canada/University Industry program (UI-11407) and La Succession J.A. DeSève. Thomas Grewal was supported by the Deutsche Forschungsgemeinschaft (DFG). We thank Dr Anne Minnich (Aventis) for helpful discussions. We are grateful to A. Chamberland, C. Lefevre, and J. Lavigne for excellent technical assistance.
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