Transcriptional Activation of Scavenger Receptor Expression in Human Smooth Muscle Cells Requires AP-1/c-Jun and C/EBPß

Both AP-1 Binding and JNK Activation Are Induced by Phorbol Esters and Oxidative Stress

From the Gladstone Institute of Cardiovascular Disease and Cardiovascular Research Institute (M.M., R.E.P.), and the Departments of Pediatrics (M.M.) and Pathology (R.E.P.), University of California, San Francisco, and the Department of Medicine (C.K.G.), University of California, San Diego.

Correspondence to Robert E. Pitas, PhD, Gladstone Institute of Cardiovascular Disease, PO Box 419100, San Francisco, CA 94141-9100. E-mail rpitas{at}gladstone.ucsf.edu

	Abstract

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Abstract—Reactive oxygen species generated by treatment of smooth muscle cells (SMCs) with either phorbol 12-myristate 13-acetate or with the combination of H₂O₂ and vanadate strongly induce expression of the class A scavenger receptor (SR-A) gene. In the current studies, cis-acting elements in the proximal 245 bp of the SR-A promoter were shown to direct luciferase reporter expression in response to oxidative stress in both SMCs and macrophages. A composite activating protein-1 (AP-1)/ets binding element located between –67 and –50 bp relative to the transcriptional start site is critical for macrophage SR-A activity. Mutation of either the AP-1 or the ets component of this site also prevented promoter activity in SMCs. Mutation of a second site located between –44 and –21 bp, which we have identified as a CCAAT/enhancer binding protein (C/EBP) element, reduced the inducible activity of the promoter in SMCs by 50%, suggesting that combinatorial interactions between these sites are necessary for optimal gene induction. Interactions between SMC nuclear extracts and the SR-A promoter were analyzed by electrophoretic mobility shift assay. c-Jun/AP-1 binding activity, specific for the –67- to –50-bp site, was induced in SMCs by the same conditions that increased SR-A expression. Moreover, phorbol 12-myristate 13-acetate, H₂O₂, or the combination of H₂O₂ and sodium orthovanadate (vanadate) activated c-Jun–activating kinase. The binding activity within SMC extracts specific for the C/EBP site was shown to be C/EBPß in SMCs. Taken together, these findings demonstrate that reactive oxygen species regulate the interactions between c-Jun/AP-1 and C/EBPß in the SR-A promoter. Furthermore, induction of oxidative stress in THP-1 cells, with a combination of 10 µmol/L vanadate and 100 µmol/L H₂O₂, induced macrophage differentiation, adhesion, and SR activity. These data suggest that vascular oxidative stress may contribute to the induction of SR-A expression and thereby promote the uptake of oxidatively modified low density lipoprotein by both macrophage and SMCs to produce foam cells in atherosclerotic lesions.

Key Words: scavenger receptor • activating protein-1 • c-Jun–activating kinase • phorbol ester • oxidative stress

	Introduction

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Circulating monocytes that have migrated into the subendothelial space terminally differentiate into macrophages with the coincident expression of numerous immediate/early and late genes. Types 1 and 2 class A scavenger receptors (SR-A) are among the regulated late genes.^{1 2 3} After differentiation, SR-A expression in macrophages is enhanced 6-fold.^{4 5} Likewise, phorbol 12-myristate 13 acetate (PMA) treatment of the monocytic cell line THP-1 results in cell differentiation, adhesion, and up to an 8-fold increase in SR-A gene transcription.⁶ SR-A expression is not limited to macrophages. Rabbit fibroblasts, as well as rabbit and human smooth muscle cells (SMCs), can be induced to express SR-A.^{7 8} Fibroblast and SMC SR-A activity is increased on incubation with platelet secretory products, certain growth factors and cytokines, PMA, and reactive oxygen species.^{9 10 11 12 13 14} In vivo, intimal SMCs express the receptor, whereas medial SMCs do not.¹³ Treatment of SMCs in vitro with either PMA or reactive oxygen increases SR-A mRNA levels, protein expression, and SR-A activity. Protein kinase C (PKC) activation by PMA induces oxidative stress within SMCs, accounting at least in part for the observed induction of SR-A activity.¹⁴ Similarly, treatment of SMCs with H₂O₂ and vanadate induces intracellular oxidative stress. Vanadate acts synergistically with H₂O₂ in many systems, promoting free-radical formation and increasing protein tyrosine phosphorylation.^{14 15 16 17} The upregulation of SMC receptor expression by oxidative stress is due to enhanced gene transcription as determined by nuclear run-on assay.¹⁴ These findings place the SR-A in a growing family of oxidative stress–response genes, which includes heme oxygenase (HO),¹⁸ metallothionein,¹⁹ collagenase,²⁰ and glutathione reductase,²¹ as well as the c-Fos and c-Jun subunits of activating protein-1 (AP-1), a redox-sensitive transcription factor involved in the induction of many of these redox-sensitive genes. Whereas we have demonstrated induction of SR-A activity in SMCs by reactive oxygen,¹⁴ similar activation of receptor expression has not been demonstrated in macrophages.

DNA footprint analyses have identified 6 sites in the SR-A genomic sequence, from -246 to +46 bp relative to the transcriptional start site, that have potential significance for SR-A expression.⁶ A composite AP-1/ets binding element located between –67 and –50 bp is critical for SR-A expression during PMA-induced macrophage differentiation.⁶ Macrophage-specific expression is conferred by an Spi-1/pu.1 binding site located at –198 to –185 bp.⁶ While the proximal 245 bp of the SR-A promoter are sufficient to confer reporter gene expression,⁶ the addition of a transcription enhancer element at –4.1 to –4.5 kb increases basal expression and confers maximal induction by PMA in macrophages in cell culture.²² These promoter elements also target in vivo expression of a reporter gene to macrophage-derived foam cells within atherosclerotic lesions of transgenic mice.²³

Here we report the results of studies to identify promoter and enhancer elements that are important for the regulation of SMC SR-A expression induced by PMA and reactive oxygen. We demonstrate that the composite AP-1/ets element is critical to the upregulation of SR-A promoter activity in SMCs. Furthermore, the specific c-Jun/AP-1 amino-terminal activating kinase (JNK) is induced in SMCs by the same conditions that increase SR-A expression. A CCAAT/enhancer binding protein (C/EBP) binding element is also necessary for full promoter activation in SMCs. These data demonstrate that redox-sensitive transcriptional control accounts for the induction of SR-A expression in SMCs under conditions that generate oxidative stress. Finally, we show that reactive oxygen can induce THP-1 monocytes to differentiate into macrophages and concomitantly induces macrophage SR-A receptor expression.

	Methods

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Materials and Cells
FBS, Dulbecco's modified Eagle's medium, Dulbecco's PBS, penicillin, streptomycin, Na¹²⁵I, and the fluorescent probe 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) were obtained as previously described.⁷ PMA, sodium orthovanadate (vanadate), and H₂O₂ were purchased from Sigma Chemical Co. Immediately before each use, the concentration of active H₂O₂ was measured and standardized by spectrophotometry on the basis of an extinction coefficient of 0.0393 at 240 nm. Primary human aortic SMCs and SM growth medium 2 were purchased from Clonetics (San Diego, Calif). The SMC lines were maintained at 37°C in 7% CO₂. THP-1 and U937 monocytic cell lines (both from American Type Culture Collection, Rockville, Md) were maintained in RPMI (Life Technologies) at 37°C in 5% CO₂. Human LDLs (d=1.02 to 1.05 g/mL) were obtained from plasma, labeled with the DiI,²⁴ and acetylated (Ac) as previously described.^{10 25}

Electromobility Shift Assays (EMSAs)
Nuclear extracts from rabbit and human SMCs, treated with either 100 nmol/L PMA or a combination of 100 µmol/L H₂O₂ and 10 µmol/L vanadate for 0.5, 1, 3, 6, 12, and 18 hours, were prepared by high-salt microextraction²⁶ and compared with nuclear extracts from untreated (control) cells. For each reaction, the salt concentration was adjusted to 100 mmol/L NaCl. Nuclear proteins (5 µg, unless otherwise specified) were preincubated at 37°C for 10 minutes with nonspecific inhibitor (1 µg of poly-dI/dC). In control samples, a 50-fold molar excess of either unlabeled AP-1 or AP-2 binding site oligonucleotide was added during the preincubation. Binding patterns were evaluated in detail for the AP-1/ets site at –67 to –50 bp and an additional hypersensitive site at –44 to –21 bp. For the initial AP-1/ets time-course reactions, a ³²P-labeled AP-1 consensus binding site oligonucleotide (Promega), labeled by T4 polynucleotide kinase phosphorylation, was used as a probe. After a preincubation period, the probe (10 000 cpm) was added to each reaction mix, and the mixture was incubated at room temperature for 20 minutes. Reactions were stopped with gel loading buffer (at a final concentration of 10% glycerol), and the complexes were resolved on 6% polyacrylamide gels. Subsequent EMSAs with AP-1–positive extracts were performed with the actual sequence of the human SR-A promoter AP-1/ets binding element (–67 to –50 bp) or mutants of either the AP-1 or ets element, respectively, as previously described.²² For supershift analyses, 200 ng of specific c-Jun, Jun-B, fos, or ets1/2 antibodies (Santa Cruz Biotechnologies) was added to the protein extract incubations after addition of the probe.

For additional binding studies, the human SR-A sequence of the –44- to –21-bp binding element plus flanking sequences was used as a probe. Competition was performed with unlabeled double-stranded oligonucleotide of the same SR-A sequence and with unlabeled C/EBP consensus wild-type (WT) and mutant binding elements. For supershift analyses, performed as above, 200 ng of specific antibodies for C/EBPß and GADD153 (Santa Cruz Biotechnologies) was used.

Endogenous JNK Kinase Assays
A pGEX4T-3 expression construct containing the amino-terminal 79 amino acids of c-Jun was kindly provided by Dr Silvio Gutkind (National Institutes of Health, Bethesda, Md). The resulting fusion protein was purified from bacterial lysates with the aid of glutathione-Sepharose beads (Pharmacia), quantified after Commassie blue staining of SDS–polyacrylamide gel electrophoresis gels by reference to known concentrations of BSA, and used as the substrate for the kinase assays.

Confluent human aortic SMCs were serum starved for 2 hours and then incubated with agonists at 37°C. At specific time points, cells were washed with PBS and lysed at 4°C in a buffer containing 25 mmol/L HEPES, pH 7.5, 0.3 mol/L NaCl, 1.5 mmol/L MgCl₂, 0.2 mmol/L EDTA, 0.5 mmol/L DTT, 20 mmol/L ß-glycerophosphate, 1 mmol/L vanadate, 1% Triton X-100, 100 µg/mL PMSF, 20 µg/mL aprotinin, and 20 µg/mL leupeptin. Cell lysates were rocked for 3 hours at 4°C in the presence of 10 µg glutathione S-transferase (GST)–c-Jun79 fusion protein bound to glutathione-agarose beads. Beads were washed 3x with PBS containing 1% Nonidet P-40 and 2 mmol/L vanadate and once with 100 mmol/L Tris, pH 7.5, and 0.5 mol/L LiCl. Samples were then resuspended in 30 µL of kinase reaction buffer containing 1 µCi [-³²P]ATP per reaction and 20 µmol/L of unlabeled ATP. After 20 minutes at 30°C, the reactions were terminated by the addition of 10 µL of SDS gel loading buffer (100 mmol/L Tris-HCl, pH 6.8, 200 mmol/L DTT, 4% SDS, 0.2% bromophenol blue, and 20% glycerol), heated at 95°C for 5 minutes, and separated by electrophoresis on 10% SDS acrylamide gels.

Plasmid Constructs and Transfection Assays
The WT human SR-A promoter sequences from –245 to +46 bp alone or with the 400-bp PMA-responsive enhancer element (–4.5 to –4.1 kb) were cloned upstream of the firefly luciferase cDNA in the expression vector 5'PSV2 luciferase.²² Promoter mutants were created by site-directed mutagenesis of the positive transcriptional elements corresponding to the Spi-1/pu.1 site at –198 to –185 bp, the AP-1 and ets components of the composite AP-1/ets element at –67 to –50 bp, and the sequence between –44 and –21 bp (a newly identified C/EBP element), as previously detailed.⁶ C/EBP site and AP-1/ets site concatemer expression constructs were created by cloning 3 consecutive sites, respectively, in tandem in front of a minimal human prolactin promoter (–69 to +33 bp) upstream of 5'PSV2 luciferase. Human SMCs were transiently transfected by using 400 µg/mL DEAE-dextran and 1.0 µg of DNA per 35-mm plate at 50% confluence. Cells were treated in duplicate with agonists 24 to 30 hours after transfection and harvested for determination of luciferase activity 16 hours after treatment. THP-1 and U937 cells were transfected by electroporation as previously described.²⁷ Low levels of background luciferase activity were subtracted before calculating the relative luciferase activity for treated versus untreated cells. All values were normalized for the total cellular protein. An extensive series of studies has been performed with ß-actin–ß-galactosidase reporter gene constructs as internal standards. There was no evidence that ß-actin and SR-A promoters influenced each other. Duplicate internal ß-galactosidase control transfections were used to confirm comparable transfection efficiencies in different experiments.

THP-1 Monocyte Differentiation Studies
THP-1 cells (10⁶) were plated in 35-mm wells in RPMI without serum and with or without PMA (100 nmol/L) or the combination of H₂O₂ (100 µmol/L) and vanadate (10 µmol/L) and incubated at 37°C. After 48 hours, the medium was removed. Cells in suspension (presumably undifferentiated) were counted, washed with PBS, resuspended in RPMI containing 10% FBS, and incubated with DiI-acetylated (Ac)LDL (5 µg/mL) overnight. The adherent cells in their original plates were also washed with PBS and incubated with fresh RPMI containing 10% FBS and 5 µg/mL DiI-AcLDL. After incubation with DiI-AcLDL, adherent cells were lifted with a cell scraper in 1x PBS. Cells were collected by centrifugation, resuspended in PBS–3% paraformaldehyde, and analyzed with a fluorescence-activated cell sorter (FACS) (cytometer model 440, Becton Dickinson) for the uptake of DiI-AcLDL, as previously described.⁷

	Results

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Effects of Mutant Promoter Elements on Basal SR-A Promoter Activity
Regulatory elements important for SMC SR-A expression were assessed by transiently transfecting SMCs with DNA constructs containing a luciferase reporter and an SR-A promoter. The sequences between –245 and +46 bp relative to the transcriptional start of the SR-A gene are sufficient to regulate SR-A activity in human THP-1 cells⁶ and SMCs (Figure 1). Including the 400-bp upstream enhancer element located between –4.1 and –4.5 kb doubled the basal activity of the –245- to +46-bp SR-A promoter in human SMCs, as reflected by the relative luciferase activity (Figure 1). The basal activity of the –245- to +46-bp SR-A promoter in human SMCs was unaffected by mutation of the Spi-1/pu.1 site, an essential element for high basal levels of SR-A activity in macrophages.⁶ Mutation of the AP-1 half of the composite AP-1/ets binding element located at –67 to –50 bp lowered the basal activity by 30%, suggesting that basal levels of AP-1 binding and activity in resting SMCs may account for some level of basal transcription. Mutation of either the ets half of this composite site or mutation of the C/EBP binding element located between –44 and –21 bp had no effect on basal promoter activity.

View larger version (17K): [in this window] [in a new window]   Figure 1. Relative transcriptional activity of WT and mutant SR-A promoter constructs in human SMCs. Minimal WT basal SR-A promoter alone is compared with promoter in the presence of the 400-bp SR-A macrophage enhancer located between –4.5 and –4.1 kb (WT+Enhancer) and with 4 minimal promoters in which regions recognized by nuclear proteins in monocyte/macrophage-derived cell lines have been mutated (as denoted by the X's): Spi-1/pu.1 site (open box), AP-1 site (hatched box), ets site (stippled box), and C/EBP site (filled box). All promoter constructs direct the expression of a luciferase reporter gene. Each was transfected in duplicate into human SMCs, and cells were examined for luciferase activity 48 hours after transfection and 15 to 16 hours after either no treatment, treatment with 100 nmol/L PMA, or treatment with the combination of 100 µmol/L H2O2 and 10 µmol/L vanadate. Results from 3 independent experiments are expressed as mean basal luciferase activity (±SD) (left) and mean ratio (±SD) of luciferase activities for treated versus untreated cells (right).

AP-1 and C/EBP Elements Are Required to Induce SR-A Promoter Activity in SMCs
Wild-type and mutant SR-A promoter and luciferase reporter constructs were subsequently evaluated in the presence of treatments known to upregulate the native SR-A gene in SMCs.¹⁴ Both PMA and reactive oxygen significantly increased transcriptional activity of the wildtype (WT) SR-A promoter (Figure 1). Mutation of the Spi-1/pu.1 element did not significantly affect responsiveness of the promoter to PMA or oxidative stress. Consistent with the critical role previously demonstrated for the AP-1/ets element in SR-A gene induction in THP-1 macrophages, mutation of either the AP-1- or ets-binding sequence within the composite site virtually abolished upregulation of the SR promoter by either treatment in SMCs (Figure 1). Interestingly, despite the importance of the AP-1/ets element within the basal SR-A promoter for maximal induction of SMC SR-A activity, the inclusion of the 400-bp upstream enhancer element with its additional AP-1/ets element did not affect the levels of induction by either treatment. In contrast, the enhancer element increased SR-A induction by PMA 4- to 5-fold in macrophages.²²

Another notable difference between SR-A regulation in macrophages and SMCs relates to the C/EBP-binding element at –44 to –21 bp. Initially identified by DNase footprinting studies with THP-1 macrophages and mouse macrophage p388D1 cell extracts, this element exhibited no significant role in either basal or inducible SR-A gene expression in macrophages.⁶ However, mutation of this site consistently reduced inducibility of SR-A expression in SMCs by PMA or reactive oxygen by 50% (Figure 1). These sequences showed only low basal activity when spliced in front of a minimal prolactin promoter in a luciferase reporter construct and were minimally upregulated in SMCs by treatment with either PMA or reactive oxygen (data not shown). This suggests that the factors binding to this sequence serve a positive regulatory function in SMCs by cooperating with AP-1 proteins.

Although the binding element at –44 to –21 bp exhibits only 60% identity with the human C/EBP consensus sequence, it closely resembles a C/EBP consensus site identified in a series of avian retroviral long-terminal repeats²⁸ (Figure 2A). We evaluated the binding of proteins within SMC nuclear extracts to the –44- to –21-bp sequence by EMSA and revealed a tripartite binding pattern. Treatment with either PMA or reactive oxygen only minimally increased the binding (Figure 2B). The binding appeared to be specific for the C/EBP family of transcription factors. It was completely inhibited by competition with WT C/EBP consensus binding site oligonucleotide but unaffected by a mutant C/EBP site oligonucleotide. The ability of a specific antibody to C/EBPß to shift the binding activity in all 3 bands suggests that this isoform accounts for most, if not all, of the observed binding activity (Figure 2C). Variable band intensities in the complex binding pattern are attributed to the differential translational and posttranslational processing characteristic of the C/EBPß trans-activator protein.²⁹

View larger version (33K): [in this window] [in a new window]   Figure 2. C/EBPß binding to –44- to –21-bp footprint of SR-A promoter. A, Human SR-A promoter element located between –44 and –21 bp is compared with consensus C/EBP-binding element and avian retroviral C/EBP consensus element. B, EMSA showing binding of human SMC nuclear protein to a C/EBP-like site. Nuclear extracts prepared from untreated human SMCs were compared with extracts prepared after 2-hour treatment with either 100 nmol/L PMA or the combination of 100 µmol/L H2O2 and 10 µmol/L vanadate (H/V), as indicated. Aliquots (5 µg) of nuclear protein were incubated with radiolabeled oligonucleotides incorporating human SR-A sequences at –44 to –21 bp. Complexes were resolved on nondenaturing 6% polyacrylamide gel run for 6 hours (free probe had exited the gel). C, EMSA showing specificity of binding of SMC nuclear protein to C/EBP-like site. Nuclear extracts prepared from untreated human SMCs were incubated with radiolabeled C/EBP-site probe, with or without 50-fold molar excess of unlabeled competitors (either consensus wt C/EBP binding site or mutant (Mut) C/EBP binding site). After a 20-minute protein-DNA binding reaction on ice, antibodies (200 ng) specific for C/EBPß or another C/EBP family member, GADD153, were added, as indicated, for an additional 20-minute incubation at room temperature. Complexes were resolved on a nondenaturing 6% polyacrylamide gel for 2 hours. A prominent and specific tripartite binding pattern is indicated by arrows. All bands were supershifted by C/EBPß antibody.

PMA or Reactive Oxygen Increases AP-1 Binding in Human SMCs
Because the AP-1/ets binding element at –67 to –50 bp is critical for the induction of SMC SR-A expression (Figure 1), we examined the induction of AP-1-binding activity in SMC nuclear extracts isolated at baseline and on induction with either PMA or reactive oxygen. Marked induction of AP-1 binding to the consensus AP-1 element was seen in human SMCs after treatment. The levels of AP-1 binding after PMA treatment were higher than those after treatment with H₂O₂ and vanadate. A 4-hour exposure of the gel containing samples treated with PMA (Figure 3, left) is compared with an overnight exposure of the gel containing samples treated with reactive oxygen (Figure 3, right). This results in greater intensity of AP-1 binding at baseline for the longer exposure. A significant increase in AP-1 binding was apparent at 1 hour in human cells treated with PMA and by 2 hours after treatment with reactive oxygen. Levels returned to baseline by 18 hours in reactive oxygen–treated cells but appeared to be sustained after PMA treatment. The binding activity was specific. In duplicate samples of extracts taken at 2 hours after treatment, binding activity was completely inhibited by a 50-fold molar excess of unlabeled AP-1-binding site but was unaffected by a 50-fold molar excess of the unrelated unlabeled AP-2-binding site.

View larger version (60K): [in this window] [in a new window]   Figure 3. EMSAs showing time-dependent changes in AP-1 levels induced in human SMCs by treatment with PMA or with the combination H2O2 and vanadate. Human SMCs were treated with 100 nmol/L PMA or 100 µmol/L H2O2 plus 10 µmol/L vanadate for the indicated times. Nuclear extracts (5-µg aliquots) were complexed with radiolabeled AP-1 consensus site probe and then resolved on 6% nondenaturing polyacrylamide gels. A 4-hour exposure of the PMA autoradiogram is compared with an overnight exposure of the H2O2/vanadate autoradiogram. Specificity of binding was confirmed by competition with a 50-fold molar excess of either an unlabeled AP-1 consensus binding site or an unrelated AP-2-binding element.

Because the human SR-A promoter AP-1-binding element differs slightly from the consensus sequence, EMSAs were repeated with the human SR-A gene AP-1/ets sequence as the probe (Figure 4). The nuclear extract isolated 2 hours after treatment with 100 µmol/L H₂O₂ and 10 µmol/L vanadate formed a specific complex with the native promoter AP-1/ets element, which was completely inhibited with a 50-fold molar excess of the unlabeled WT promoter element. There was no inhibition by unlabeled oligonucleotide in which the AP-1 half of the composite site had been mutated. However, a 50-fold molar excess of unlabeled oligonucleotide in which the ets component of the composite AP-1/ets site was mutated competed for the binding as effectively as the WT sequence. Consistent with these competition studies, the AP-1 mutant/WT ets site, when labeled as the probe, formed no complex, whereas the WT AP-1/ets mutant site as the probe gave a complex similar to the WT promoter element. These studies confirm that AP-1 accounts for all of the observed binding activity at the AP-1/ets site.

View larger version (89K): [in this window] [in a new window]   Figure 4. EMSAs showing AP-1 binding to human SR WT and mutant (Mut) AP-1/ets binding elements. Human SMC nuclear extracts prepared after a 2-hour treatment with 100 µmol/L H2O2 and 10 µmol/L vanadate were complexed with WT AP-1/ets (–67 to –50 bp) sequence from human SR-A promoter. Specificity of protein-DNA complex was confirmed by competition with unlabeled WT AP-1/ets element and lack of competition by AP-1 mutant/ets binding element. Unlabeled AP-1/ets mutant binding element competed as effectively as the WT sequence. Ability of c-Jun antibody to supershift the complex indicates that the principal AP-1 subunit involved in this interaction is c-Jun. Consistent with competition experiments, there is no protein-DNA binding when the AP-1/ets probe is mutated at the AP-1 site, but a normal complex forms when the probe is mutated at the ets site.

The AP-1-binding activity was partially supershifted by addition of c-Jun antibody, suggesting that this is a principal component of the observed AP-1-binding activity (Figure 4). Antibodies to Jun-B or fos had no effect on the binding complex, nor did an antibody that recognizes both ets1 and ets2 (data not shown). Taken together, these data suggest that treatment of SMCs with either PMA or reactive oxygen induces c-Jun/AP-1 binding. An ets protein likely cooperates with AP-1 on this composite element in vivo owing to the effect of the mutation in the ets site on the response to PMA and reactive oxygen. The relevant protein may be present in much lower amounts than AP-1 factors or may be degraded during extract preparation.

Both PMA and Reactive Oxygen Species Activate JNK
Because c-Jun is a component of the AP-1-binding activity identified in SMCs, we evaluated the possibility that it was regulated posttranslationally by H₂O₂ and vanadate. c-Jun is activated by phosphorylation of serines at positions 63 and 73 in the amino-terminal activation domain by a specific kinase, JNK.³⁰ JNK is itself activated by threonine and tyrosine phosphorylation.³¹ Endogenous JNK activity, as indicated by the ability of treated SMC extracts to phosphorylate a c-Jun substrate fusion protein, increased 4-fold 15 minutes after treatment with 100 nmol/L PMA (Figure 5). The activity began to decrease at 30 minutes and had nearly returned to baseline by 60 minutes. In a separate experiment, 100 µmol/L H₂O₂ alone induced even higher levels of JNK activity: a 10-fold increase at 15 minutes, which was sustained at a lower level (4-fold) at 30 minutes and returned to baseline by 60 minutes. Vanadate (10 µmol/L) alone had no appreciable effect. Consistent with previously reported synergism of H₂O₂ with vanadate on SR-A induction in SMCs,¹⁴ the combination of 10 µmol/L vanadate and 100 µmol/L H₂O₂ increased JNK activity 11-fold at 15 minutes, increasing to 12-fold at 30 minutes, and falling more slowly back to baseline by 90 minutes.

View larger version (26K): [in this window] [in a new window]   Figure 5. Induction of c-Jun kinase activity in human SMCs. Confluent plates of human SMCs were deprived of serum for 2 hours and then treated in serum-free medium for the times indicated with 100 nmol/L PMA or with 100 µmol/L H2O2, 10 µmol/L vanadate, or both. Untreated cells maintained for 2 hours in serum-free medium were used as controls. Cell number was used to normalize the amount of extract used in this assay. Cells were lysed, and kinase activity in cell extracts was recovered by mixing with kinase substrate bound to glutathione-agarose beads (10 µg of GST–c-Jun79 recombinant fusion protein), prepared as described in Methods. After 3 hours, beads were spun down, washed, and incubated in kinase buffer containing [-32P]ATP for 20 minutes at 30°C. Phosphorylated proteins were resolved by electrophoresis on 10% SDS-polyacrylamide gels. Position of GST–c-Jun79 is indicated.

Oxidative Stress Induces THP-1 Monocyte-to-Macrophage Differentiation and SR-A Gene Expression
The SR-A gene is constitutively expressed at high levels in macrophages. Macrophage internalization of oxidized lipoproteins is thought to contribute to cholesterol engorgement and foam cell formation in atherosclerotic lesions.¹ Because the induction of SMC SR-A expression is mediated in part by reactive oxygen,¹⁴ we next investigated whether oxidative stress per se could be linked to SR-A gene expression in the macrophage. Treatment of THP-1 cells with the combination of 10 µmol/L vanadate and 100 µmol/L H₂O₂ for 48 hours induced up to 50% of monocytes to become adherent. Approximately two thirds of the adherent cells actively took up DiI-AcLDL, as reflected in a 62-fold increase in mean fluorescence compared with control cells (Figure 6). PMA treatment was somewhat more effective. Up to 90% of the cells treated for 48 hours with PMA became adherent, of which 80% manifested increased DiI-AcLDL uptake, reflected in a 70-fold increase in mean fluorescence (Figure 6). There was no uptake of DiI-AcLDL in nonadherent cells (data not shown). The monocyte-to-macrophage differentiation and the increase in uptake of DiI-AcLDL were correlated with specific SR-A transcriptional upregulation. Because the AP-1/ets element is critical for receptor expression in both SMCs and macrophages, this site was concatemerized in front of a minimal prolactin promoter and luciferase reporter gene. When this construct was transfected into THP-1 cells, treatment with either PMA or a combination of 10 µmol/L vanadate and 100 µmol/L H₂O₂ significantly increased luciferase activity (Figure 6c). The relative fold induction in macrophages by PMA is twice that seen with H₂O₂ and vanadate, as previously observed in SMCs with the full SR-A promoter (Figure 1). Similar results were observed in a second monocyte cell line, U937 (data not shown). Taken together, these data establish that reactive oxygen species exert a potent regulatory function in the expression of the SR-A gene in macrophages as well as SMCs.

View larger version (49K): [in this window] [in a new window]   Figure 6. Monocyte-to-macrophage differentiation and induction of SR-A activity on treatment with reactive oxygen. A, Phase-contrast (panels 1 and 3) and fluorescence (panels 2 and 4) photomicrographs of THP-1 macrophages that have differentiated, become adherent, and taken up fluorescently labeled DiI-AcLDL after 48 hours of treatment with the combination of 10 µmol/L vanadate and 100 µmol/L H2O2 (panels 1 and 2) or 100 nmol/L PMA (panels 3 and 4). Control (untreated) cells neither adhered nor took up DiI-AcLDL (data not shown). B, Quantification of monocyte-to-macrophage differentiation by FACS analysis. THP-1 cells treated as described in (A) were lifted by cell scraping, resuspended in 1x PBS–3% paraformaldehyde, and examined by FACS. FACS analyses for untreated control cells (hatched line) and cells treated with either PMA (dotted line) or H2O2 and vanadate (H/V) (solid line) are superimposed. Uptake and internalization of DiI-AcLDL (increase in relative fluorescence intensity) indicate SR-A activity. C, AP-1/ets site (–67 to –50 bp) concatemerized (x3) in front of a minimal prolactin promoter/luciferase reporter was transfected in triplicate into THP-1 monocytic cells. Cells were examined for luciferase activity 48 hours after transfection and 15 to 16 hours after either no treatment (control) or treatment with 100 nmol/L PMA or with 100 µmol/L H2O2 plus 10 µmol/L vanadate alone (Van) or in combination (H/V). Results are expressed as mean fold induction over control (±SD) of luciferase activity.

	Discussion

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Expression of the SR-A gene was first demonstrated in cells of the monocyte/macrophage lineage. We subsequently demonstrated that SR-A expression can be induced in SMCs in response to PMA, certain growth factors, and oxidative stress.^{7 12 14} Here we have demonstrated that transcriptional activation of the SR-A gene in macrophages and SMCs depends on overlapping but nonidentical sets of transcription factors. SR-A gene expression in macrophages is critically dependent on Spi-1/pu.1, a B-cell– and macrophage-restricted transcription factor.^{6 23} Not surprisingly, the pu.1-binding site was not required for SR-A promoter activity in SMCs, consistent with the lack of pu.1 expression in this cell type. In contrast, a binding site in the SR-A promoter for C/EBP, which is not of functional importance in macrophages,⁶ was essential for full promoter activity in SMCs stimulated with PMA or reactive oxygen. Induction of SR-A expression in SMCs by PMA or the combination of H₂O₂ and vanadate was dependent on the composite binding site for AP-1 and cooperating ets domain transcription factors, located between –50 and –67 bp relative to the major transcriptional start site. Increases in SMC c-Jun/AP-1 binding and JNK activity and in C/EBP binding in response to PMA or reactive oxygen have been demonstrated. Independent work has shown upregulation of SMC ets-1 mRNA expression by factors known to regulate vascular SMC migration and proliferation, including phorbol esters.³²

In macrophages, AP-1 and ets are downstream targets of the macrophage colony-stimulating factor (M-CSF) receptor, which regulates macrophage proliferation and differentiation by coupling to components of several signal transduction pathways, including the Ras/Raf/mitogen-activated protein (MAP) kinase cascade. Mutations of the AP-1/ets-binding sites in the SR-A promoter and upstream enhancers abolish transcriptional responses to M-CSF in transgenic mice, indicating that these sites are of critical importance for the regulation of SR-A gene expression in vivo (F. Guidez and C.K. Glass, unpublished observations, 1997). The treatment of resident peritoneal macrophages with M-CSF has been associated with "priming" the oxidative burst.³³ Although M-CSF receptors are not expressed by normal vascular SMCs, high-affinity binding of M-CSF with the resultant increase in tyrosine phosphorylation has been demonstrated in intimal SMCs isolated from a rabbit model of atherosclerosis.³⁴ Increased tyrosine phosphorylation is associated with increased intracellular oxidative stress.³⁵ H₂O₂ activates receptor tyrosine kinase–mediated signaling events directly in vascular SMCs, leading to Ras activation.³⁶ The current studies suggest that activation of Ras by oxidative stress may mimic receptor-dependent signaling in both SMCs and macrophages and is sufficient to activate the SR-A gene in both cell types. This promiscuous activation of the MAPK pathway could potentially contribute to cholesterol accumulation in atherosclerotic lesions.

SMC SR-A expression has been demonstrated in rabbit atherosclerotic lesions.¹³ We have demonstrated that SMC SR-A expression in stimulated rabbit SMCs is 3-fold higher than in human SMCs.¹⁴ Low levels of SR-A gene expression make its detection difficult in human lesions but do not preclude relevance of the SR-A in vivo in a disease process that evolves over decades. Furthermore, our studies demonstrate the existence of an oxidative stress–response pathway in SMCs that results in the activation of AP-1 proteins and is likely to be relevant to the expression of multiple inflammatory SMC genes. Finally, we extended our analysis of the oxidative stress pathway to a human monocytic leukemia cell line, which has been a model for the study of SR-A gene expression in macrophages.

The AP-1-binding element within the proximal SR-A promoter is essential for induction of SR-A gene activity in both macrophages and SMCs. AP-1 has long been known to mediate gene induction by PMA.³⁷ AP-1 transcriptional activity has since been found to be induced by numerous growth factors and cytokines. Many of the factors that induce AP-1 activity³⁸ have been independently shown to induce intracellular oxidative stress, including PMA,^{14 39} platelet-derived growth factor,⁴⁰ transforming growth factor-ß,⁴¹ tumor necrosis factor-, and interleukin-1.⁴² These factors have also been associated with the induction of SMC SR-A activity.^{7 12 13} Our previous observation, that direct treatment of either human or rabbit SMCs with reactive oxygen species was sufficient to induce SR-A expression,¹⁴ led us to hypothesize that reactive oxygen serves as a common denominator in the mediation of SR-A upregulation by numerous factors in atherosclerotic lesions. Duplicate copies of an AP-1/ets composite element closely resembling the sequence between –67 and –50 bp of the human SR-A promoter have been identified between –981 and –769 bp of the intercellular adhesion molecule-1 gene promoter. Consistent with our observations, this site has also been identified as an H₂O₂-responsive element.⁴³

c-Jun contributes significantly to the observed AP-1-binding activity induced in SMCs by either PMA or reactive oxygen treatment (Figure 4). Although c-Jun expression is rapidly induced by extracellular signals, its posttranslational activity is tightly regulated by protein phosphorylation.³⁰ PKC activation decreases phosphorylation of c-Jun by casein kinase II at sites that negatively regulate its activity,^{44 45} and we have recently shown that PKC plays a role in SMC SR-A upregulation.¹⁴ However, this alone only partially accounts for c-Jun activation. c-Jun phosphorylation by JNK (the specific c-Jun–activating kinase) at 2 serines within the c-Jun trans-activation domain is required for maximal transcriptional activity.^{46 47 48} JNK, a serine/threonine kinase, is itself activated by dual threonine/tyrosine phosphorylation.³¹ The activation of JNK by several forms of cellular stress, including UV irradiation, heat shock, and inflammatory cytokines, has resulted in the description of the JNK subgroup of MAPKs as "stress-activated" protein kinases.⁴⁹ The potent induction of JNK activation by UV light strongly suggests that oxidative stress mediates its activation, as UV exposure causes lipid peroxidation and decreases the level of reduced glutathione in cells.⁵⁰ The UV response, like the response to oxidative stress, is characterized by the induction of tyrosine kinase activity via the inhibition of tyrosine phosphatases.⁵¹ The sensitivity of protein tyrosine phosphatases to reactive oxygen species has been attributed to a critical conserved cysteine residue that is vulnerable to oxidative modification.⁵² JNK activation in turn results from unopposed tyrosine kinase activity. Consistent with this scheme, reactive oxygen species were recently directly implicated in JNK activation by cytokines.⁵³ Our current results demonstrate that the same conditions associated with increased AP-1 binding and SR-A gene expression within SMCs also activate JNK, providing the first direct link between this stress-activated MAPK and SR-A regulation.

In addition to AP-1/ets factors, the functional significance of an additional site within the basal promoter at –44 to –21 bp has led to the identification of cooperative involvement of C/EBPß in SMC SR-A transcription. Although the –44- to –21-bp element conforms only loosely to the consensus C/EBP-binding site, it closely resembles elements identified within avian retroviral long-terminal repeats that were found to bind C/EBP specifically (Figure 2A). The C/EBP family is composed of at least 3 major members designated as , ß, and , characterized by the presence of a conserved domain in the carboxyl-terminal region of the protein. C/EBP isoforms are implicated in regulating processes relevant to cellular proliferation, differentiation, and expression of cell type–specific genes.⁵⁴ Although C/EBP expression is restricted to the liver and adipose tissues, C/EBPß and are widely expressed. We demonstrate in this report that a tripartite protein-DNA interaction involving the SR-A promoter sequences at –44 to –21 bp can be attributed to C/EBPß (Figure 2). The intron-less gene of the C/EBPß trans-activator protein encodes a single mRNA, which in turn results in several related proteins,²⁹ which may explain the complex binding pattern observed. The various isoforms have been attributed to differential initiation and inhibition of translation at specific AUG sites within the mRNA.⁵⁵ Various prooxidants, including lipopolysaccharide, cytokines,⁵⁶ and heavy metals,⁵⁷ have been associated with increased expression, phosphorylation, and binding of C/EBPß, with resultant induction of several acute-phase genes. There is precedent for coordinate regulation by adjacent AP-1- and C/EBP-binding elements in the promoter of another redox-sensitive gene. A 268-bp promoter fragment that mediates basal level and inducer-dependent activation of the mouse HO-1 gene contains 2 AP-1 sites with 2 C/EBP-binding sites directly upstream. Site-directed mutagenesis of 1 or more of the C/EBP and AP-1 sites revealed that each of the 4 elements is required for optimal activity of the enhancer fragment.⁵⁷

The enhancement of gene expression by oxidative stress is likely to represent a protective physiological response. Activated macrophages produce high levels of superoxide via NADPH oxidase activity, which results in a variety of reduced oxygen metabolites that are potent bactericidal and tumoricidal agents. Yet the macrophages themselves survive. At least 2 other gene products induced in both macrophages and SMCs by oxidative stress are involved in mediating important cytoprotective responses to free-radical generation, HO-1, and mouse stress-inducible 23-kDa protein (MSP23).^{58 59} In the case of HO-1, it has been speculated that gene induction leads to a reduction in the cellular pool of heme and heme-containing proteins, thereby removing potential prooxidant catalysts.⁶⁰ Furthermore, the end product of the heme degradation pathway mediated by HO-1 is bilirubin, which has intrinsic antioxidant properties.⁶¹ MSP23 has recently been shown to belong to a new type of thiol-specific antioxidant family, which protects glutamine synthetase from inactivation by a mixed metal-thiol oxidation. HO-1 and MSP23 appear to be part of a coordinate antioxidant cellular response.⁵⁹ Upregulation of SR-A activity by oxidative stress may also be consistent with cellular self-defense. Innate immune defense systems, like the SR-A, use cell-surface proteins to recognize toxic substances according to their particular carbohydrate structures.⁶² The fact that bacterial lipopolysaccharide is a ligand for SR-A,⁶³ as well as the recent demonstration of increased susceptibility to infection in the SR-A knockout mouse model,⁶⁴ supports a protective physiological role for this receptor. Oxidatively modified LDL in atherosclerotic lesions contributes to vascular oxidative stress^{65 66} and to the induction of numerous inflammatory genes that contribute to atherosclerosis.⁶⁷ Oxidized LDL also increase SR-A gene expression in SMCs (preliminary data not shown). In the context of excess oxidized lipoproteins, however, the "protective" removal of these proinflammatory cytotoxins by SRs appears to represent a 2-edged sword, which results in foam cell formation and atherogenesis.

	Acknowledgments

 
This work was supported in part by the National Institutes of Health program project grant No. HL47660 (to R.E.P.). We thank September Plumlee for manuscript preparation; Gary Howard and Stephen Ordway for editorial support; and Amy Corder, Stephen Gonzales, and John Carroll for photography and graphics. FACS analysis was performed by Dr Eric Weider in the Gladstone Institute of Virology and Immunology.

Received December 29, 1997; accepted March 27, 1998.

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