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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:969-978.)
© 1997 American Heart Association, Inc.

Articles

Regulation of Scavenger Receptor Expression in Smooth Muscle Cells by Protein Kinase C

A Role for Oxidative Stress

Michele Mietus-Snyder; Annabelle Friera; Christopher K. Glass; ; Robert E. Pitas

From the Gladstone Institute of Cardiovascular Disease (M.M., A.F., R.E.P.), the Cardiovascular Research Institute (M.M., R.E.P.), and the Departments of Pediatrics (M.M.) and Pathology (R.E.P.), University of California at San Francisco, and the Division of Cellular and Molecular Medicine and Endocrinology and Metabolism (C.K.G.), University of California at San Diego, La Jolla.

Correspondence to Robert E. Pitas, PhD, Gladstone Institute of Cardiovascular Disease, PO Box 419100, San Francisco, CA 94141-9100.

	Abstract

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Abstract Phorbol esters increase scavenger-receptor mRNA expression and receptor activity in smooth muscle cells (SMCs). Our present results demonstrate that activation of protein kinase C (PKC) mediates this increase in receptor expression. This conclusion is based on the findings that (1) phorbol esters induced translocation of PKC- from the cytosol to the membrane fraction; (2) PKC inhibitors blocked the effect of phorbol esters on receptor expression; (3) diacylglycerol, a physiological PKC agonist, enhanced scavenger-receptor activity; and (4) in cotransfected human SMCs, constitutively active PKC- stimulated the expression of a reporter gene under control of the scavenger-receptor promoter. Phorbol ester treatment of SMCs increased intracellular reactive oxygen, and the increase in receptor activity was reduced 30% by the antioxidant N-acetyl cysteine (NAC), suggesting a role for reactive oxygen in phorbol ester–mediated receptor regulation. Furthermore, direct treatment of SMCs with reactive oxygen species increased scavenger-receptor activity. In rabbit SMCs, 100 µmol/L H₂O₂ alone slightly increased scavenger-receptor mRNA and protein expression. In combination, 100 µmol/L H₂O₂ and 10 µmol/L vanadate, which promotes formation of OH and enhances the inhibition of protein tyrosine phosphatase by H₂O₂, increased scavenger-receptor mRNA expression 25-fold in rabbit SMCs and 8-fold in human SMCs. NAC reduced the effect of H₂O₂ and vanadate by 93%. The increase in SMC scavenger-receptor expression occurs at the level of gene transcription. Receptor mRNA half-life was unchanged after treatment with either phorbol esters or reactive oxygen (14.5 hours), and induction by phorbol esters increased SMC scavenger-receptor mRNA transcription, as determined by nuclear run-on assay. Multiple cytokines and growth factors that contribute to the generation of reactive oxygen species are present in atherosclerotic lesions. These factors may all contribute to the upregulation of SMC scavenger-receptor activity and therefore to the formation of smooth muscle foam cells.

Key Words: scavenger receptor • receptor regulation • reactive oxygen • protein kinase C

	Introduction

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Two alternatively spliced (type I and type II) class A¹ scavenger-receptor cDNAs have been cloned from bovine, human, and murine macrophages.^{2 3 4 5} In SMCs, these receptors exhibit properties essentially identical to those of the types I and II macrophage scavenger receptors.^{6 7 8 9} The class A scavenger receptors are constitutively expressed at high levels in macrophages, and their internalization of oxidized lipoproteins may contribute to cholesterol engorgement and formation of macrophage foam cells in atherosclerotic lesions.^{10 11} We have proposed a similar mechanism to explain the lipid accumulation in intimal SMCs.^{6 7}

Whereas SMCs in the arterial intima of cholesterol-fed New Zealand White rabbits express scavenger receptors, SMCs in the arterial media do not.¹² These observations suggest that factors in the developing atherosclerotic lesion regulate receptor activity. Receptor expression can also be regulated in vitro. Phorbol esters significantly increase the normally low levels of scavenger-receptor expression in cultured human and rabbit SMCs.^{9 13 14} We have recently shown that growth factors present in atheroma and associated with c-fms (a characteristic gene of monocyte/macrophages) expression in SMCs¹⁵ act synergistically to upregulate SMC scavenger receptors.¹³ The growth factors with the most potent individual effects on SMC scavenger-receptor expression, PDGF and transforming growth factor-ß₁,¹³ induce moderate production of ROS in cells.^{16 17 18} Phorbol esters also generate superoxide and H₂O₂ in cells through PKC-mediated activation of NADPH oxidase.¹⁹

Along with numerous other metabolic effects, ROS regulate gene expression.²⁰ The association of ROS with both PKC- and growth factor–mediated metabolic pathways suggests a role for ROS in regulation of the scavenger-receptor gene. Here, we show that treatment of SMCs with phorbol esters results in the translocation and activation of PKC, an increase in intracellular ROS, and an increase in scavenger-receptor gene transcription and receptor activity. Oxidative stress induced by treatment of rabbit and human SMCs with H₂O₂ and vanadate also increases class A scavenger-receptor mRNA expression and activity. The increase in SMC scavenger-receptor expression after treatment with phorbol esters is mediated, in part, by ROS.

	Methods

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Materials
FBS, DMEM, PBS, penicillin, streptomycin, [¹²⁵I]NaI, the fluorescent probe DiI, and rabbits were obtained as previously described.⁹ The PKC inhibitors H-7 and MDL29,152 were, respectively, purchased from Sigma and a gift from Marion Merrell Dow (Cincinnati, Ohio). The diacylglycerol kinase inhibitor R59022 was obtained from Calbiochem. DAG, PMA, NAC, catalase, sodium orthovanadate (vanadate), and H₂O₂ were purchased from Sigma. The concentration of H₂O₂ was measured by spectrophotometry, based on an extinction coefficient of 0.0393 at 240 nm, and standardized immediately before each use. DCFH-DA was purchased from Molecular Probes, and isotype-specific PKC- antibody was obtained from Life Technologies.

Lipoproteins
Human LDLs (d=1.02 to 1.05 g/mL) were obtained from plasma, labeled with the fluorescent probe DiI,²¹ and acetylated as previously described.^{7 22} The LDLs were iodinated as described.²³

Cells
The New Zealand White rabbit SMC line SMC-2 was provided by Drs Lisa Minor and George Rothblat (Medical College of Pennsylvania). A spontaneously transformed rabbit SMC line, Rb-1, was provided by Dr Murice Naachtigal (University of South Carolina). Both were maintained in DMEM containing 10% heat-inactivated FBS. Primary human aortic SMCs and medium (Sm-GM2) were purchased from Clonetics. The SMC lines were maintained at 37°C in 7% CO₂.

Plasmid Constructs and Transfection Assays
A wild-type human scavenger-receptor promoter–luciferase reporter construct, which contains scavenger-receptor promoter sequences (-245 to +46 bp), subcloned upstream of the firefly luciferase cDNA in the expression vector D5'PSV2 luciferase,²⁴ was obtained from Dr Christopher Glass (University of California at San Diego). Expression vectors for wild-type PKC- and a constitutively active PKC- mutant were obtained from Dr Masa-aki Muramatsu (University of Tokyo).²⁵ Human SMCs were transiently cotransfected in duplicate with equal amounts of the reporter construct and either the wild-type or the constitutively active PKC- expression vector by use of 400 µg/mL of DEAE/dextran and 2.0 µg of total DNA per 35-mm plate at 50% cell confluency. For determination of luciferase activity, cells were harvested 48 hours after transfection. Low background levels of luciferase activity (determined in cells transfected with an unrelated DNA) were subtracted before the relative luciferase activity for each transfection was calculated. All values were normalized for total cellular protein content. Duplicate ß-galactosidase control transfections were used to confirm comparable transfection efficiencies among different experiments.

Lipoprotein Degradation and Internalization Analysis
For degradation studies, cells were incubated in medium as described above with various agonists or inhibitors as described in the figure legends. The cells were then washed with DMEM, ¹²⁵I-Ac-LDL (5 µg/mL) was added, and the cells incubated for 16 hours at 37°C. The medium was centrifuged to remove cells, and scavenger-receptor activity was assayed by measurement of the trichloroacetic acid–soluble lipoprotein degradation products in the medium, as described.²⁶ Nonspecific degradation, defined as the amount of degradation obtained in the presence of a 100-fold excess of unlabeled ligand, was subtracted from all data. The cellular protein content was determined by analysis of the cells solubilized in 0.1N NaOH, with BSA used as the standard.²⁷

Internalization of DiI-Ac-LDL by cells was quantified by FACS analysis. Cells were incubated with agonists or inhibitors in serum-free medium at 37°C for 1.5 hours, washed, and incubated for 16 to 24 hours with agonist-free full-growth medium. DiI-labeled lipoproteins (5 µg/mL) were added, and the cells were incubated for 8 hours at 37°C. The cells were removed from the tissue culture dishes with trypsin-EDTA, suspended at a concentration of 1x10⁶ cells/mL in PBS containing 3% paraformaldehyde, and analyzed with a fluorescence-activated flow cytometer (model 440, Becton-Dickinson) as previously described.⁹

Flow Cytometric Detection of Reactive Oxygen Production in SMCs
Reactive oxygen production in SMCs was detected by measurement of the free radical–induced conversion of DCFH (nonfluorescent) to 2'7'-dichlorofluorescein (DCF) (fluorescent) by FACS analysis as previously described.²⁸ DCFH-DA diffuses freely through cell membranes and is immediately hydrolyzed by intracellular esterases to DCFH, which is trapped within cells. Free radicals, particularly OH·, convert DCFH to DCF, which can be measured by FACS. SMCs were trypsinized and suspended at 5x10⁵ cells/mL in basal medium (EBM, Clonetics) without phenol red. All analyses were performed within 45 minutes of trypsinization. Cells in suspension at 37°C were treated with agonists for various times; DCFH-DA (5 µm) was added 15 minutes before FACS analysis. The increase in fluorescence detected at 515 to 555 nm was corrected for spontaneous conversion of DCFH to DCF in control cells treated identically but without agonist.

RNA Isolation and Northern Analyses
Total RNA was isolated from cultured cells using the monophasic phenol–guanidine isothiocyanate TRIzol reagent (Life Technologies). Northern blot analyses were performed with either total or poly A–containing RNA. Samples were electrophoresed on 1% agarose gels containing 2.2 mol/L formaldehyde, transferred to nylon membranes, and cross-linked by UV irradiation. The filters were prehybridized for 2 hours, followed by an overnight hybridization, all at 42°C in 50% formamide solution. The rabbit SMC RNA was hybridized to a cDNA fragment from the collagen-like domain, and to control for any variation in RNA loading, membranes were stripped and rehybridized with a rabbit actin probe. The human mRNA was initially hybridized with a species-specific cDNA probe from the coiled-coil domain and then stripped and rehybridized with a cDNA fragment from the GAPDH gene. The cDNA probes were prepared by PCR-based amplification of the scavenger-receptor cDNAs with primers generated from published sequences^{4 8} spanning the collagen-binding domain for the rabbit and the -helical coiled-coil domain in the human. The 5'- and 3'-oligonucleotides used correspond to rabbit nucleotides 764 to 783 and 1010 to 1041 and human nucleotides 589 to 618 and 869 to 893, respectively. All probes were Klenow-labeled with [-³²P]dCTP (3000 Ci/mmol, DuPont/NEN). To differentiate type I and type II rabbit scavenger-receptor mRNA by Northern blot analysis, type-specific probes were generated. The type I–specific probe prepared by PCR amplification corresponded to the type I receptor carboxy-terminus (1042 to 1365 bp). A type II–specific 60-mer probe was synthesized on the basis of the published sequence of the 3' untranslated region (1074 to 1133 bp) and hybridized to poly A RNA purified from rabbit peritoneal macrophages with oligo(dT)cellulose columns (Boehringer Mannheim). The relative scavenger-receptor mRNA intensity was quantified by phosphorimage densitometry.

Immunocytochemistry
The SMCs were seeded at 1x10⁴ cells/well in an eight-well chamber slide. Immunocytochemistry was performed essentially as described.²⁹ When the cells were 90% confluent, treatment was started and continued for 24 hours. Cells were washed in PBS and fixed with 3% paraformaldehyde. After permeabilization and preincubation for 30 minutes at room temperature in blocking solution (0.1% milk, 150 mmol/L ammonium acetate, 0.15% Triton X-100, and 1:50 dilution of normal horse serum, prepared in 0.5xPBS), the cells were incubated for 1 hour at 4°C with an antibody (from Dr Mason Freeman, Massachusetts General Hospital, Harvard Medical School) raised to a synthetic peptide corresponding to the first 17 amino acids of the amino-terminus (cytoplasmic domain) of the rabbit scavenger receptor.¹² Cells were washed three times in wash buffer (same as blocking solution but with 0.1% Triton X-100 and 1xPBS) and then incubated for 30 minutes at room temperature with a 1:4000 dilution of biotinylated anti-goat IgG secondary antibody (Vector Laboratories) in the same buffer. Endogenous peroxidase activity was inhibited by incubating cells with 0.3% H₂O₂ in methanol for 30 minutes. Cells were then incubated for 30 minutes at room temperature with an avidin-biotinylated horseradish peroxidase complex (Vector Laboratories). Reactive cells were visualized by a 2-minute incubation in diaminobenzydine solution (Vector Laboratories), washed, and counterstained for 5 minutes with 1% methyl green in H₂O.

RNA Half-life and Nuclear Run-on Experiments
To determine mRNA half-life, replicate plates of rabbit SMCs were either untreated, treated with 65 nmol/L PMA, or treated with a combination of 10 µmol/L vanadate and 100 µmol/L H₂O₂ for 1.5 hours. After 14 hours, when maximal scavenger-receptor mRNA levels are observed after induction (data not shown), actinomycin D was added to the plates at a concentration of 5 µg/mL. Total RNA was isolated at multiple time points (0, 1.5, 9, 12, and 18 hours) as described above. RNA samples (15 µg) were loaded onto a nylon filter by use of a dot blot apparatus and cross-linked by UV irradiation. After hybridization with a rabbit scavenger-receptor mRNA-specific probe, signals were quantified by phosphorimage analysis and densitometry.

For the nuclear run-on experiment, nuclei were prepared from 5x10⁷ SMCs treated with 65 nmol/L PMA or vehicle. RNA transcripts were synthesized and labeled as described.³⁰ Plasmid DNA containing the rabbit type II scavenger-receptor cDNA (a gift from Dr Mason Freeman) and a 179-bp fragment from the rabbit actin gene (5 µg each) were linearized, bound to nitrocellulose filters with a slot blot apparatus, and hybridized to the labeled nascent RNA as described.³⁰

Protein Kinase C Translocation and Enzymatic Assays
Human SMCs were grown to confluence in 100-mm plates in full-growth medium (Sm-GM2, Clonetics) and then incubated for 15 minutes in basal medium alone (SMBM, Clonetics) or in basal medium containing 65 nmol/L PMA or the combination of 10 µmol/L vanadate and 100 µmol/L H₂O₂. Cells from five plates were used for each treatment. Cells were rinsed with PBS, scraped in extraction buffer (20 mmol/L Tris-HCl [pH 7.5], 0.5 mmol/L EDTA, 0.5 mmol/L EGTA, and 25 µg each of aprotinin and leupeptin), and Dounce homogenized. The homogenates were fractionated by centrifugation at 100 000g for 30 minutes, and the supernatants (cytosolic fraction) were retained. The pellets (membrane fraction) were resuspended in extraction buffer plus 0.5% Triton X-100, Dounce homogenized, and recentrifuged at 100 000g for 30 minutes. The resulting supernatants containing solubilized membrane proteins were retained. Both cytosolic and membrane protein fractions were applied to DEAE Sephacel columns; washed with 20 mmol/L Tris-HCl [pH 7.5], 0.5 mmol/L EDTA, 0.5 mmol/L EGTA, and 10 mmol/L ß-mercaptoethanol and eluted in 2 mL of the same buffer containing 200 mmol/L NaCl. Enzymatic assays for PKC activity were performed immediately as described below. The remaining samples were divided into aliquots for protein determination and stored at -70°C for subsequent Western blotting.

Enzymatic activity was determined with a PKC assay kit (Life Technologies), which measures the phosphorylation of a synthetic peptide from myelin basic protein, as described.³¹ Briefly, protein extracts were incubated for 20 minutes at room temperature with either a PKC activation preparation or a pseudo–substrate inhibitor peptide, and the incorporation of [-³²P]ATP into myelin basic protein was measured. Activity was calculated as total picomoles PKC per minute and normalized for cell number. For Western blot analysis, cytosolic and membrane fraction proteins were separated by electrophoresis on 10% polyacrylamide gels and transferred to nitrocellulose. Membranes were blocked for 1 hour with 1% BSA in TBST followed by a 30-minute incubation with a PKC- isotype–specific antibody (Life Technologies) at 1.5 µg/mL. The blot was washed once in TBST containing 0.5 mol/L NaCl followed by two washes with regular TBST, each for 20 minutes. Donkey anti-rabbit antibody conjugated to horseradish peroxidase (Amersham) was then added at a 1:8000 dilution in TBST and incubated at room temperature for 30 minutes, followed by three 20-minute washes in TBST. Detection was performed by enhanced chemiluminescence with an ECL kit (Amersham).

	Results

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Scavenger-Receptor Induction by Phorbol Esters Is Mediated by PKC
We have previously shown that the induction of SMC scavenger-receptor activity by phorbol esters is associated with an increase in receptor mRNA expression, protein levels, and receptor activity.⁷ However, the mechanism by which these occur has not been determined. Induction of scavenger-receptor activity by phorbol esters suggests a role for PKC, because phorbol esters mimic the activity of DAG, a physiological PKC activator. To explore this possibility more fully, SMCs were treated with DAG to activate PKC directly or with phorbol esters in the presence of PKC inhibitors. Because DAG is rapidly converted to phosphatidic acid by the action of DAG kinase, both single and multiple additions of DAG were evaluated as well as the addition of DAG in the presence of a DAG kinase inhibitor, R59,022.³² Incubation of SMCs with 25 to 50 µmol/L DAG for 3 hours increased scavenger-receptor activity threefold (Fig 1). Multiple additions of DAG or the addition of DAG together with the DAG kinase inhibitor increased scavenger-receptor activity sixfold. Further evidence that PKC plays a role in the phorbol ester–mediated induction of scavenger-receptor activity was obtained with PKC inhibitors. The PMA-induced increase in scavenger-receptor activity was blocked by both H7 and MDL29,152 (a more selective PKC inhibitor) (Fig 2).³³ The MDL29,152, which inhibited PMA induction of receptor activity by 93%, was more effective than H7.

View larger version (26K): [in this window] [in a new window]   Figure 1. Treatment of SMCs with DAG increases scavenger-receptor activity. Rabbit SMCs were treated with either a single addition of DAG (3 hours) with three successive additions of DAG at 1-hour intervals or with a single 3-hour addition of DAG in the presence of the DAG kinase inhibitor R59,022 (5 µmol/L). Cells were then washed, and full-growth medium was replaced. Scavenger-receptor activity was assayed 24 hours later by measurement of ability of cells to degrade 125I-acetyl-LDL (see "Methods"). Data presented are representative of two separate experiments.

View larger version (22K): [in this window] [in a new window]   Figure 2. Ability of PKC inhibitors to block effect of phorbol esters on SMC scavenger-receptor activity in rabbit SMCs. SMCs were preincubated for 2 hours with various concentrations of protein kinase inhibitor H7 or MDL29,152 before addition of 65 nmol/L PMA for a subsequent overnight (15-hour) coincubation. Scavenger-receptor activity was determined by measurement of ability of cells to degrade 125I-Ac-LDL (see "Methods"). This experiment was performed twice with similar results.

Thus far, all experiments with phorbol esters had been performed after an overnight incubation.^{6 7 9} Long-term incubation with PMA reduces PKC levels. If the upregulation of receptor expression is in fact PKC mediated, a relatively short incubation should also stimulate receptor activity. Twenty-four hours after a 1-hour incubation of SMCs with PMA, receptor activity was increased sixfold. In fact, Western blotting of SMC proteins extracted after only 15 minutes of PMA treatment revealed a clear shift of PKC- from the cytosolic to the membrane fraction (Fig 3). In two separate assays of enzymatic activity, 64% and 93% of the total PKC activity, respectively, shifted to the membrane fraction. By comparison, only 20% of PKC activity was found in the membrane fractions of control (untreated) cells in both experiments (data not shown).

View larger version (66K): [in this window] [in a new window]   Figure 3. Western blot showing translocation of PKC- after treatment of human SMCs. Cytosolic (C) and membrane (M) extracts were isolated from human SMCs without treatment (none) or after a 15-minute treatment with 65 nmol/L PMA. Total protein (30 µg) was loaded in each lane. Representative results from one of three independent protein extractions are shown.

To further document a direct role for PKC in SMC scavenger-receptor induction, SMCs were cotransfected with a scavenger-receptor promoter–luciferase reporter construct and expression vectors for either wild-type or constitutively active PKC- (PKC- is the principal phorbol ester–sensitive PKC isotype in SMCs³⁴ ). Cells were assayed for luciferase activity 36 to 48 hours after transfection. The wild-type PKC- construct did not increase luciferase expression, whereas cotransfection with the constitutively active PKC- mutant increased luciferase activity approximately fivefold (Fig 4).

View larger version (15K): [in this window] [in a new window]   Figure 4. Relative luciferase activity in human SMCs transfected with a human scavenger-receptor promoter–luciferase reporter construct24 (SR-Luc) or cotransfected with SR-Luc and a vector expressing wild-type PKC- or one expressing a constitutively active mutant PKC- (PKC AC).25 Cells were transfected in duplicate and tested for luciferase activity 48 hours later. Luciferase activity is reported compared with that observed with SR-Luc alone. Results are expressed as the mean±SD of three independent experiments.

Scavenger Receptor Induction by ROS in Rabbit and Human SMCs
PMA induces NADPH oxidase,¹⁹ which results in the generation of free radicals. To determine whether oxygen radicals might be involved in the PMA-mediated regulation of scavenger-receptor activity in SMCs, we examined the effect of the antioxidant NAC on receptor regulation. We found that NAC reduced phorbol ester induction of receptor activity by 35%. Since ROS have been implicated in other PKC-mediated pathways,^{35 36} we evaluated more directly the ability of ROS to stimulate scavenger-receptor activity in rabbit and human SMCs. Because exogenously added H₂O₂ is rapidly metabolized by antioxidant enzymes, the effects of H₂O₂, with and without vanadate, on scavenger-receptor mRNA expression were evaluated. Sodium orthovanadate acts synergistically with H₂O₂ in many systems,^{37 38 39} undergoing conversion to pervanadate, which can promote free radical formation via activation of NADPH oxidase^{40 41} and increase ROS-mediated protein tyrosine phosphorylation. To determine whether enhancement of scavenger-receptor activity by vanadate could result from increased generation of endogenous ROS, the generation of free radicals in SMCs was analyzed by flow cytometric detection of DCF fluorescence as an indirect measure of ROS production in cells (Fig 5). In the absence of agonist, there was minimal change in the concentration of DCF in the cells at 30 seconds, 5 minutes, or 15 minutes after the addition of vehicle (Fig 5A). Treatment with vanadate alone did not differ from control. Addition of 100 µmol/L H₂O₂ induced an immediate (30 seconds) and marked but short-lived (<5 minutes) increase in intracellular ROS (data not shown). The effects of pervanadate and phorbol esters were then evaluated. Pervanadate was generated as previously described⁴² by incubation of H₂O₂ and vanadate for 15 minutes followed by treatment with 200 µg/mL catalase to destroy residual free H₂O₂ before the addition to SMCs. Any DCF conversion should therefore reflect the production of intracellular ROS. Phorbol esters (Fig 5B) and pervanadate (Fig 5C) both gave a more gradual but sustained increase in DCF conversion, increasing free radicals at 5 minutes and generating measurable oxidative stress through the 15-minute time point.

View larger version (37K): [in this window] [in a new window]   Figure 5. FACS analysis showing generation of reactive oxygen in SMCs. Human SMCs were trypsinized and suspended in basal medium without phenol red at a concentration of 1x106 cells/mL. Cells were loaded with DCFH-DA (5 µmol/L) at 37°C for 15 minutes before treatment. Cells were treated with agonist, and serial FACS analyses were begun immediately (time 0) and repeated at indicated time points. Each panel represents the overlay of four FACS analyses after addition of no agonist (A), 65 nmol/L PMA (B), or 10 µmol/L pervanadate (C).

The effects of these treatments on scavenger-receptor mRNA expression were assessed by Northern blot analysis. Rabbit and human SMCs were treated with phorbol ester for 1.5 hours. Total RNA was isolated 24 hours after treatment and subjected to Northern analysis. Cells exposed to phorbol esters displayed higher levels of scavenger-receptor mRNA than nontreated control cells (Fig 6A and 6B). The addition of 100 µmol/L H₂O₂ to SMCs for 1.5 hours mildly increased scavenger-receptor mRNA levels in rabbit SMCs compared with the control cells. H₂O₂ or sodium orthovanadate alone had little effect on scavenger-receptor expression in human SMCs. However, 100 µmol/L H₂O₂ and 10 µmol/L sodium orthovanadate had a synergistic effect. Phosphorimage densitometry showed that rabbit and human SMC scavenger-receptor mRNA levels were increased 6-fold and 4.3-fold, respectively, to the levels obtained after phorbol ester treatment. Although comparable inductions in mRNA were obtained in both rabbit and human cells, the absolute levels of receptor mRNA were 8 to 10 times greater in rabbit SMCs. The reasons for this difference are being investigated.

View larger version (36K): [in this window] [in a new window]   Figure 6. Northern blot analysis showing scavenger-receptor mRNA from rabbit and human SMCs incubated with PMA or ROS. Northern blots of total RNA (15 µg) from rabbit (A) and human aortic (B) SMCs treated as indicated. Scavenger-receptor mRNA was detected by hybridization with species-specific cDNA probes that recognize both type I and type II receptors (see "Methods"). Lower panels of A and B show hybridization of same blots with control probes for rabbit actin and human GAPDH mRNAs, respectively. C, Poly A–containing rabbit peritoneal macrophage RNA (3 µg) hybridized with a collagen-like domain probe common to type I and type II scavenger receptors (left lane) reveals a banding pattern identical to that seen with induced SMCs. Preferential hybridization to lower and upper bands with probes specific for type I (middle lane) and type II (right lane) receptors, respectively, is shown.

We observed three distinct class A scavenger-receptor mRNA species, which were similar in size and relative intensity to those previously described for rabbit SMCs and rabbit peritoneal macrophage scavenger-receptor mRNA (Fig 5A and References 7⁷ and 12¹² ). Although the hybridization patterns for receptor mRNA are indistinguishable, the total yield of scavenger-receptor RNA is >100-fold greater from peritoneal macrophages than from induced SMCs. For this reason, we used poly A RNA prepared from peritoneal macrophages for receptor type–specific Northern analyses. Hybridization with probes specific for either the type I or type II receptor demonstrated that the smaller mRNA species encoded the type I message, whereas the larger species represented type II mRNA transcripts (Fig 6C). These results are in agreement with previous analysis of the murine scavenger-receptor mRNA transcripts.⁴³ Both receptor variants are clearly upregulated by phorbol ester or ROS treatment (Fig 6A and 6B).

Immunocytochemistry experiments confirmed that the increase in scavenger-receptor mRNA induced in PMA- or ROS-treated SMCs was associated with an increase in scavenger-receptor protein (Fig 7). Scavenger-receptor immunoreactivity to a rabbit scavenger-receptor– specific antibody was higher in rabbit SMCs treated with PMA, H₂O₂, or H₂O₂ and vanadate than in control cells.

View larger version (104K): [in this window] [in a new window]   Figure 7. Photomicrographs showing enhanced scavenger-receptor expression in rabbit SMCs after treatment with PMA or ROS. Rabbit SMCs were either untreated (A) or treated with 65 nmol/L PMA (B), 100 µmol/L H2O2 (C), or combination of 10 µmol/L vanadate and 100 µmol/L H2O2 (D) for 1.5 hours and analyzed 24 hours later. Immunocytochemistry performed with a rabbit scavenger-receptor antibody (see "Methods") showed more scavenger-receptor protein in treated cells than in control cells.

To demonstrate that the ROS-mediated increase in receptor expression was associated with an increase in receptor activity, human and rabbit SMCs were incubated with H₂O₂ in the presence or absence of vanadate, and the internalization of DiI-Ac-LDL was assessed by FACS analysis (Fig 8). In rabbit and human SMCs, receptor activity was relatively unchanged by the addition of either 100 µmol/L H₂O₂ or 10 µmol/L vanadate alone. In rabbit SMCs, however, there was clear upregulation with 200 µmol/L H₂O₂. Vanadate and H₂O₂ together had a synergistic effect, increasing scavenger-receptor activity 25-fold in rabbit SMCs and 8-fold in human SMCs. This level of receptor induction was similar to that obtained with phorbol esters in rabbit SMCs and was approximately half that in phorbol ester–treated human SMCs (Fig 8). The absolute levels of receptor activity in human and rabbit SMCs differed substantially. In rabbit SMCs treated with H₂O₂ and vanadate, the relative fluorescence intensity of the cells after incubation with DiI-Ac-LDL was 150 to 200. In human SMCs, it was 25 to 50. Therefore, the absolute level of receptor activity was approximately 4-fold to 5-fold greater in rabbit than in human SMCs. These data are consistent with the lower level of scavenger-receptor mRNA in human than in rabbit SMCs (Fig 5).

View larger version (18K): [in this window] [in a new window]   Figure 8. FACS analysis showing effects of PMA or H2O2, with and without vanadate, on uptake of DiI-Ac-LDL by rabbit and human SMCs. Cells were incubated for 1.5 hours with agonists indicated; 16 to 24 hours after initiation of treatment, DiI-Ac-LDL (5 µg/mL) and cells were incubated for 8 hours at 37°C. Cells were then trypsinized, fixed, and assayed for scavenger-receptor activity by FACS analysis. Data for rabbit (A) and human (B) SMCs are reported as increase in fluorescence intensity of treated cells relative to that of control cells incubated in absence of agonists. Results represent mean±SD of three independent experiments.

Preincubation of the SMCs with the antioxidant NAC (25 mmol/L) almost completely blocked (89% and 93% reductions in two separate experiments) the ROS-stimulated uptake of DiI-Ac-LDL, consistent with the hypothesis that receptor expression is increased by regulatory pathways mediated by oxidative stress. Treatment of human SMCs with 100 µmol/L H₂O₂ and 10 µmol/L vanadate, which together stimulate receptor expression, did not result in membrane translocation of PKC. In two separate assays, 80% of PKC activity (the same as in untreated control cells) remained in the cytosolic fraction of ROS-treated cells, and immunoreactive PKC, visualized by Western blot, remained in the cytosolic fraction after ROS treatment (data not shown). The lack of PKC translocation to the membrane after ROS treatment supports the hypothesis that free radicals may enter the SMC scavenger-receptor regulatory pathway downstream of PKC.

SMC Scavenger-Receptor Upregulation Results From Increased Gene Transcription
To evaluate whether increased gene transcription or protein synthesis was required to obtain the increased scavenger-receptor activity, inhibition experiments were performed with actinomycin D and cycloheximide. Both inhibition of mRNA synthesis with actinomycin D (1 µmol/L) and inhibition of protein translation with cycloheximide (at 10 µmol/L and 100 µmol/L) blocked the phorbol ester–induced upregulation of scavenger-receptor activity (Fig 9). Gene transcription and protein translation are required for the observed increase in scavenger-receptor activity. Here (Fig 6) and previously,⁷ we have demonstrated an increase in the level of scavenger-receptor mRNA after PMA treatment of SMCs. Because steady-state mRNA levels represent a balance between rates of transcription and degradation, these variables were evaluated in the presence or absence of agonist. Nuclear run-on experiments demonstrated an increase in transcription of scavenger-receptor mRNA 6 hours after treatment with PMA (Fig 10A). Run-on experiments were not performed with ROS-treated cells. However, scavenger-receptor mRNA half-life was similar in PMA-treated (t_½14.1 hours), ROS-treated (t_½15.5 hours), and untreated (t_½14.4 hours) rabbit SMCs, demonstrating that receptor mRNA half-life was relatively stable after induction with either agonist (Fig 10B). These data suggest that the induction of scavenger-receptor gene expression by phorbol esters or ROS occurs at the level of gene transcription.

View larger version (23K): [in this window] [in a new window]   Figure 9. Effect of inhibition of transcription or translation on phorbol ester–induced upregulation of scavenger-receptor activity in rabbit SMCs. Rabbit SMCs were incubated for 15 hours with medium alone (control) or with medium containing 65 nmol/L PMA alone or together with actinomycin D or cycloheximide. Inhibitors were added to cells 2 hours before addition of PMA. Effect on induction of rabbit SMC scavenger-receptor activity was assessed by measuring degradation of 125I-Ac-LDL, as described in "Methods." Data are representative of two separate experiments.

View larger version (50K): [in this window] [in a new window]   Figure 10. Scavenger-receptor mRNA gene transcription and half-life. Results of a nuclear run-on transcription assay (A). 32P-labeled mRNA was transcribed in vitro in isolated cell nuclei (5x107) from PMA-treated (6 hours) and untreated cells and hybridized to plasmid cDNAs immobilized on nylon membranes. The scavenger-receptor plasmid used contains rabbit type II scavenger-receptor cDNA, and the positive control plasmid contains 179 bp corresponding to bases 21 to 200 of rabbit actin cDNA. For mRNA half-life determination (B), rabbit SMCs received no treatment () or a 1.5-hour treatment with PMA (, 65 nmol/L) or reactive oxygen species (ROS) (, 100 µmol/L H2O2 and 10 µmol/L vanadate). Cells were then incubated for 14 hours in the absence of agonist, before addition of 5 µg/mL actinomycin D, to inhibit further transcription. RNA was isolated at times indicated after addition of actinomycin D. Total RNA (25 µg from untreated control cells and 12.5 µg from ROS- or PMA-treated cells) was applied with a slot blot apparatus and hybridized to a labeled probe specific for rabbit scavenger-receptor mRNA. Resultant signals were quantified by phosphorimage densitometry. Similar results were obtained in two separate experiments.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
We have previously shown that scavenger-receptor activity in SMCs is induced by phorbol esters.⁹ In the present studies, we have shown that phorbol esters induce the translocation of PKC- from the cytosol to the membrane fraction of human SMCs; that inhibition of PKC blocks the effect of phorbol esters on receptor expression; that DAG, a physiological PKC agonist, induces scavenger-receptor activity; and that in human SMCs, a constitutively active PKC- upregulates the expression of a reporter construct under the control of the scavenger-receptor promoter. The upregulation of scavenger-receptor activity by phorbol esters was partially blocked by the antioxidant NAC, and treatment with phorbol esters increased intracellular ROS, suggesting a role for ROS in the phorbol ester–mediated induction of scavenger-receptor activity. This hypothesis is supported by the fact that treatment of rabbit and human SMCs with ROS increased scavenger-receptor mRNA levels, protein expression, and receptor activity. While H₂O₂ alone elicits an increase in SMC scavenger-receptor expression in rabbit SMCs, the combination of H₂O₂ and vanadate is more effective in both rabbit and human SMCs. We have shown that this combined treatment leads to intracellular ROS production, presumably through the induction of NADPH oxidase by pervanadate (References 40⁴⁰ and 41⁴¹ and Fig 5). NAC reduced the ROS-mediated increase in scavenger-receptor activity. Since treatment of cells with H₂O₂ and vanadate does not result in membrane translocation of PKC activity, ROS induction of receptor activity apparently involves signaling pathways downstream of PKC. Finally, we have demonstrated that the increase in receptor activity induced by phorbol esters is blocked by inhibition of DNA transcription and that scavenger-receptor mRNA half-life was unchanged after induction by either phorbol esters or ROS. Induction by phorbol esters increased SMC scavenger-receptor mRNA transcription as determined by nuclear run-on assay. We conclude that both PMA and ROS increase SMC scavenger-receptor activity and mRNA levels by increasing gene transcription.

These data suggest that the scavenger-receptor gene belongs to the family of "stress-response genes," which encode a diverse group of acute-phase reactant proteins. Whether activated by infection, ionizing radiation, or oxidative stress, most stress-response genes (including c-fos, c-jun, tumor necrosis factor-, interleukin-1, the metallothioneins, heme oxygenase, and PKC itself) are also responsive to phorbol esters and are therefore activated via a PKC-mediated pathway.⁴⁴ Interestingly, the gene encoding heme oxygenase is also highly induced by oxidative stress and has been hypothesized to play a protective role by reducing the heme pools necessary for generating hydroxyl radicals during sustained oxidant stress.⁴⁵ Adding the scavenger-receptor gene to this list of immunologically relevant genes would be consistent with the host defense role proposed for macrophage scavenger receptors.^{46 47 47A}

Reactive oxygen species trigger PKC enzymatic activity in other cell types.^{35 39 48} We could not detect any direct membrane translocation or activation of PKC in SMCs after ROS treatment, although transient translocation and some degree of activation cannot be ruled out. The data demonstrate the generation of free radical species by phorbol ester induction of PKC. This observation is consistent with work showing that the activation of PKC in segments of rabbit aorta, by either phorbol ester or lysophosphatidyl choline, which is found in oxidized LDL, significantly enhances superoxide production by SMCs of the arterial media.⁴⁹

The mechanism whereby oxidative stress induces scavenger-receptor gene transcription is suggested by the synergism between H₂O₂ and vanadate in increasing scavenger-receptor activity. In the present studies, vanadate was used to enhance the generation of intracellular oxidative stress necessary to induce SMC scavenger-receptor expression. H₂O₂, though freely permeable, is rapidly metabolized in cell culture, and vanadate alone traverses cell membranes very poorly. However, vanadate enhances the metabolism of H₂O₂ to the more reactive hydroxyl (OH·) radical.³⁹ Furthermore, in the presence of H₂O₂, vanadate is oxidized to pervanadate,³⁷ greatly facilitating its entry into cells and the induction of intracellular free radical generation via activation of NADPH oxidase.^{38 40} The production of superoxide would be expected to result in further hydroxyl radical generation in aqueous solution by dismutation of two superoxide molecules into H₂O₂ and O₂. Hydrolysis of H₂O₂ in the presence of heat, ionizing radiation, or catalytic metals in turn generates the hydroxyl radical.⁵⁰ We have shown that the treatment of SMCs with pervanadate leads to an increase in intracellular ROS. There is increasing evidence that vanadate, which is found in vivo at concentrations of 0.3 to 1.0 µmol/L,⁵¹ plays important biological roles.⁵² Vanadate and H₂O₂ individually enhance protein tyrosine phosphorylation by specifically inhibiting protein tyrosine phosphatase activity.^{37 53} Inactivation is presumably mediated by oxidation of a highly conserved cysteine identified in a family of protein tyrosine phosphatases.⁵⁴ This inactivation of protein tyrosine phosphatase by H₂O₂ and vanadate in cells is most clearly demonstrated when the two are used in combination.^{37 38}

In the scavenger-receptor activation cascade, there are multiple steps in which enhanced protein tyrosine phosphorylation may elicit an effect. ROS may facilitate the phosphorylation of the receptor tyrosine kinases for several growth factors that mediate the regulation of scavenger-receptor activity in SMCs.^{13 14} Receptor tyrosine kinases activate phospholipase C, which in turn initiates the mitogen-activated protein kinase signaling pathway at the point of ras activation. This leads ultimately to the activation of nuclear protein factors, specifically nuclear factor-B and c-jun/AP-1.⁵⁵

The ROS-mediated induction of AP-1, which is composed of homodimers of the proto-oncogene c-jun product or heterodimers of jun and fos subunits,⁵⁶ may be particularly relevant to scavenger-receptor gene expression. Several independent lines of investigation have demonstrated increased transcription of c-jun in the presence of ROS,^{57 58} and both basal expression and phorbol ester–mediated induction of the human scavenger-receptor promoter in transiently transfected THP-1 cells depend on DNA elements that recognize AP-1 family members.^{24 59} Furthermore, ROS may play a significant role in c-jun/AP-1 protein activation. Although phosphorylation of two serine residues in the c-jun transactivation domain has been implicated in its activation, the responsible mitogen-activated protein kinase activity, c-jun amino-terminal kinase, is itself activated by tyrosine phosphorylation.⁶⁰ We have shown both that AP-1 is increased by ROS treatment of SMCs and that it is required for the expression of a scavenger-receptor promoter–luciferase reporter construct in SMCs (M. Mietus-Snyder, C.K. Glass, and R.E. Pitas, unpublished observations, 1995). Although we demonstrated comparable inductions of scavenger-receptor expression and activity in both rabbit and human SMCs, absolute levels were four to five times higher in rabbit SMCs, the more atherosclerosis-susceptible species. The reason for this difference is unknown. It could be related to differences in the level of the transcription factor AP-1. In human SMCs, the basal AP-1 level and the levels after induction are lower than in rabbit SMCs (M. Mietus-Snyder, C.K. Glass, and R.E. Pitas, unpublished observations, 1995).

Although we added ROS to the cells exogenously, numerous factors stimulate the endogenous production of ROS through PKC activation of NADPH oxidase– and xanthine oxidase–dependent pathways. Increased scavenger-receptor activity has been demonstrated in SMCs incubated with phorbol esters,⁹ cytokines,¹² and various growth factors, including PDGF and transforming growth factor-ß₁^{13 14} and in SMCs infected with cytomegalovirus.⁵⁶ These factors induce oxygen radical production in fibroblasts,^{61 62} osteoblasts,¹⁷ and, most recently, vascular SMCs (References 18¹⁸ and 63⁶³ and Fig 4), and a role for H₂O₂ in PDGF-mediated signal transduction in SMCs has been demonstrated.¹⁸ Our present results suggest a common pathway whereby multiple inflammatory cytokines and growth factors, which have been implicated in atherogenesis, may converge in the ras-mediated activation of immediate and early response genes, leading downstream to scavenger-receptor gene regulation.

	Selected Abbreviations and Acronyms

 

Ac = acetylated AP-1 = activating protein-1 DAG = diacylglycerol (1-oleoyl-2-acetyl-sn-glycerol) DCFH-DA = 2',7'-dichlorofluorescein diacetate DiI = 1,1'-dioctadecyl-3,3,3',3'-tetramethylindo-carbocyanine perchlorate FACS = fluorescence-activated cell sorter MDL29,152 = 4-propyl-5-[quinolinyl]-2[3H]-oxazolone NAC = N-acetyl cysteine PCR = polymerase chain reaction PDGF = platelet-derived growth factor PKC = protein kinase C PMA = phorbol 12-myristate 13-acetate ROS = reactive oxygen species SMC = smooth muscle cell TBST = 50 mmol/L Tris-HCl [pH 7.5], 150 mmol/L NaCl, 0.05% Tween 20

	Acknowledgments

 
This work was supported by National Institutes of Health Program Project Grant HL-47660. We thank James McGuire for excellent technical assistance; Dr Courtney Broaddus for her counsel and assistance with the DCFH assays; Don Haumant and Susannah White for manuscript preparation; Gary Howard and Stephen Ordway for editorial support; and Amy Corder, John Carroll, and Stephen Gonzales for graphics and photography. FACS analysis was performed by Kris Kavanaugh (Laboratory for Cell Analysis, University of California, San Francisco).

Received February 15, 1996; accepted September 12, 1996.

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