Participation of ATP7A in macrophage mediated oxidation of LDL.

ATP7A primarily functions to egress copper from cells, thereby supplying this cofactor to secreted copper-accepting enzymes. This ATPase has attracted significant attention since the discovery of its mutation leading to human Menkes disease and the demonstration of its distribution in various tissues. Recently, we reported that ATP7A is expressed in the human vasculature. In the present study, we investigated the cellular expression of ATP7A in atherosclerotic lesions of LDL receptor (-/-) mice. Subsequently, we examined the role of ATP7A in regulating the oxidation of LDL in a macrophage cell model. We observed that ATP7A is expressed in atherosclerotic murine aorta and colocalizes with macrophages. To investigate the function of ATP7A, we downregulated ATP7A expression in THP-1 derived macrophages using small interfering RNA (siRNA). ATP7A downregulation attenuated cell-mediated oxidation of LDL. Moreover, downregulation of ATP7A resulted in decreased expression and enzymatic activity of cytosolic phospholipase A(2) alpha (cPLA(2)alpha), a key intracellular enzyme involved in cell-mediated LDL oxidation. In addition, cPLA(2)alpha promoter activity was decreased after downregulation of ATP7A, suggesting that ATP7A transcriptionally regulates cPLA(2)alpha expression. Finally, cPLA(2)alpha overexpression increased LDL oxidation, which was blocked by coadministration of ATP7A siRNA oligonucleotides. These findings suggest a novel mechanism linking ATP7A to cPLA(2)alpha and LDL oxidation, suggesting that this copper transporter could play a previously unrecognized role in the pathogenesis of atherosclerosis.

(Stoughton, MA). LDL was stored in the dark at 4°C and used within 1 month of isolation. Prior to each experiment, the LDL was dialyzed with Slide-A-Lyzer cassettes (10,000 molcular weight cutoff; Pierce, Rockford, IL) against 1 l of phosphate-buffered saline (pH 7.4, two changes) for 24 h at 4°C, then against 0.5 l of Ham's F12 medium for 12 h. After dialysis, LDL was sterilized by passing through a low protein binding fi lter (0.45 µm pore size, Nalgene).

Thiobarbituric acid assay
The thiobarbituric acid (TBA) assay was used to assess the extent of cell-mediated LDL oxidation as described previously ( 19 ). TBA reacts with malondialdehyde (MDA) and MDA-like derivatives to form TBA reactive species, which can be quantifi ed by spectrophotometry at 535 nm. Data are presented as MDA equivalents (nM MDA/mg LDL protein).

Small interfering RNA
The ATP7A small interfering (siRNA) oligonucleotides (Ambion, Austin, TX) targeted exon 11 (sense, GCAACUAUUGUAACUC-UUGtt; antisense, CAAGAGUUACAAUAGUUGCtt). Scrambled and GAPDH siRNA sequences were used as negative controls. All siRNAs were obtained in lyophilized, annealed form, resuspended in double-distilled water to a stock concentration of 20 µM, and stored at Ϫ 20°C in 100 µl aliquots. Cells were transfected with siRNA oligonucleotides using oligofectamine transfection reagent (Invitrogen, Carlsbad, CA) or DMRIE-C reagent (Invitrogen) according to the manufacturer's instructions.

Western blotting
Cells were incubated for 30 min at 4°C in 1% Triton X-100 lysis buffer (50 mmol/L HEPES, 50 mmol/L NaCl, 5 mmol/L EDTA) with a tablet of protease inhibitors (Roche, Indianapolis, IN). Samples were then centrifuged at 18,000 g for 10 min, and proteins in the supernatant were separated using SDS-PAGE, transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA), blocked, and incubated overnight at 4°C with ATP7A or cPLA 2 ␣ primary antibodies (Santa Cruz) ( 8,19 ). After incubation with HRP-conjugated secondary antibodies, proteins were detected by chemiluminescence (Bio-Rad). Equal gel loading was determined by Ponceau S staining of nitrocellulose membrane following transfer and by blotting with ␣ -tubulin antibodies (Sigma).

RNA preparation
TRI Reagent (Sigma) was used to isolate total RNA following the manufacturer's instructions, with minor modifi cations. For total RNA isolation, after the ethanol precipitation step in the TRI Reagent extraction procedure, an additional cleanup was performed using RNeasy Mini kit (QIAGEN, Valencia, CA) to improve the purity of total RNA. In some cases, the quality of RNA was assessed using standard techniques, including examination of the 260 to 280 nm optical density absorbance ratio and detection of distinct 28S and 18S rRNA bands on ethidium bromide-stained agarose gels.

RT-PCR
cDNA was synthesized using a Retroscript First-Strand Synthesis Kit (Ambion) following the manufacturer's instructions. PCR was performed using the Mx3000P TM PCR system (Stratagene, La Jolla, CA) under the following conditions: denaturation at 94°C for 1 min, annealing at 55°C for 30 s, and extension at 72°C for 1 min. All RT-PCR experiments were performed in triplicate. Equal aliquots from 25 or 30 thermocycles were electrophoresed in 1.5% agarose gel and quantifi ed by densitometry analysis (Kodak Digital 1D Science). The abundance of target mRNA was calculated in relation to the GAPDH mRNA in the same sample. ATP7A might play a previously unrecognized role in the regulation of atherosclerosis.
In the present study, we investigated the cellular expression of ATP7A in murine atherosclerotic lesions. Subsequently, we examined the role of ATP7A in regulating LDL oxidation in a macrophage cell model. Our study also revealed a potential novel link between ATP7A, cPLA2 ␣ , and LDL oxidation. Based on these studies, we conclude that ATP7A is expressed in macrophages and may function to facilitate LDL oxidation.

Animal studies
Eight-week-old LDL receptor (LDLR) Ϫ / Ϫ mice on a C57BL/6 background (Jackson Laboratories, Bar Harbor, ME) were maintained on a Western-type high-fat/-cholesterol diet for 12 weeks and then euthanized. The heart and aorta were perfused with phosphate-buffered saline. The aortic root and ascending aorta were carefully dissected and placed in phosphate-buffered saline containing 30% sucrose for 18 h, embedded in OCT medium, frozen, and sectioned with a cryostat at 10 µm intervals throughout the aortic sinus and the aortic arch. Following immunostaining with antibodies against ATP7A and MOMA ( 8, 21 ), sections were viewed under an Olympus fl uorescence microscope with excitation at 455/490 nm, a band pass of 500 nm, and a 515 nm barrier fi lter. Equal exposure time was used for all the images.

Cell culture
The human monocytic cell line THP-1 was obtained from the American Type Culture Collection (Rockville, MD) and maintained in culture using RPMI 1640 medium (Hyclone, Logan, UT) supplemented with 10% FBS, penicillin (100 units/ml), and streptomycin (100 µg/ml). Cells were maintained in 5% CO 2 tissue culture incubators at 37°C until ready for use. Cells were stimulated by 100 nM PMA (Sigma, St. Louis, MO) to induce differentiation into macrophages. The differentiated THP-1 cells, hereafter referred to as THP-1-derived macrophages, have previously been used as a physiologically robust macrophage model as evidenced by the expression of cytokines and adhesion molecules, differentiation markers, and phagocytic function characteristic of macrophages ( 22,23 ). For experiments to assess LDL oxidation, THP-1-derived macrophages were incubated with native LDL (250 µg/ml protein) in Ham's F12 medium without FBS for the indicated time periods.

Human LDL preparation
LDL was prepared from the serum of human volunteers by discontinuous density gradient ultracentrifugation. In some experiments, LDL was purchased from Biomedical Technologies current report, immunofl uorescence was performed to determine the cell type(s) in which ATP7A was expressed in vivo. Figure 1 illustrates representative data showing that ATP7A expression is largely colocalized with expression of MOMA, a widely used mouse macrophage subpopulation marker, in intimal lesions of aortas from LDLR Ϫ / Ϫ mice. The specifi city of our antibody was verifi ed by demonstrating lack of ATP7A expression in fi broblasts isolated from a patient with Menkes disease ( 32 ), whereas expression was detected in fi broblasts isolated from a healthy control patient (data not shown). Therefore, these data suggest that ATP7A is expressed in macrophages within atherosclerotic lesions.

Inhibition of LDL oxidation after downregulation of ATP7A in THP-1-derived macrophages
To begin to investigate the function of ATP7A in macrophages, siRNA technique was employed to downregulate the expression of ATP7A in THP-1-derived macrophages. THP-1 cells can be induced by PMA to differentiate into a macrophage-like cell, as defined by an increase in adherence and surface expression of macrophage specific markers. Following treatment with 100 nM PMA for 24 h to induce macrophage differentiation, the THP-1 cells were incubated with siRNA oligonucleotides to downregulate ATP7A expression. Figure 2A shows that after treatment with ATP7Aspecific siRNA oligonucleotides for 24 h, ATP7A protein was downregulated by ≈ 9-fold compared with that of scrambled oligonucleotides and nonoligonucleotide treated cells. Moreover, this level of inhibition of ATP7A cPLA 2 ␣ primer sequence: forward: TGGCTCTGTGTGATCAG-GAG, reverse: GAGCCAGAAAGACCAGCAAC. GAPDH primer sequence: forward: AACACAGTCCATGCCATCAC, reverse: TCC-ACCACCCTGTTGCTGTA. cPLA 2 activity assay cPLA 2 activity was determined using an assay kit (Cayman Chemical) with 2-deoxy-2-thioarachidonoylphosphatidylcholine as the substrate, as described previously ( 24 ). To exclude secretory PLA 2 and calcium-independent PLA 2 , supernatants of cell homogenates were concentrated by Y30 fi lters (Millipore), followed by incubation with bromoenol lactone (Cayman Chemical), a calcium-independent PLA 2 inhibitor. Samples (10 µl) were fi nally assayed in a 96-well plate, and the OD values were measured at 414 nm.

Cell transfection and reporter assays
A cPLA 2 ␣ reporter construct containing 2.4 kb ( Ϫ 2,487 to +40 bp) of a 5 ′ -fl anking region of rat cPLA2 ␣ gene was ligated into the promoterless vector PA3-Luc (PA3-Luc/cPLA2 ␣ ) ( 25,26 ). The phRLTK vector (Promega) containing the Renilla luciferase gene was used as an internal control. Transfections of THP-1 cells were carried out using DMRIE-C reagent (Invitrogen) as previously described ( 27 ). The DMRIE-C reagent was fi rst mixed with 5 µg of PA3-Luc/cPLA 2 ␣ vector DNA, 0.5 µg of phRLTK vector, and 5 nM of control or ATP7A siRNA oligonucleotides to form DMRIE-C-DNA complexes. After cells were transfected with DMRIE-C-DNA complexes in 200 µl of serum-free Opti-MEM I (Invitrogen) for 4 h, 2 ml of growth medium containing 15% FBS and 100 nM PMA was added to each well for an additional 48 h incubation. Cells were then harvested, and cell lysates were extracted with passive lysis buffer (Promega), which was used to determine the luciferase activity. The luciferase activity was then normalized to Renilla luciferase activity and expressed as relative luciferase activity.

Transfection of cPLA 2 ␣ plasmids
A human cPLA 2 ␣ construct was cloned into the enhanced cyan fl uorescent protein (ECFP) vector (Clotech) kindly provided by Dr. Christina C. Leslie, National Jewish Medical and Research Center ( 28,29 ). Overexpression of cPLA 2 ␣ using this plasmid mimics the function and cellular translocation of the endogenous enzyme (29)(30)(31). DMRIE-C reagent was used to transfect ECFP-cPLA 2 ␣ vector DNA (4 µg) and siRNA oligonucleotides (5 nM) into THP-1 cells. Four hours after transfection, 2 ml of growth medium containing 15% FBS and 100 nM PMA was added into each well for an additional 48 h. Expression of ECFP-cPLA 2 ␣ was monitored by fl uorescence microscopy and further confi rmed by Western blot.

Statistical analyses
Data are presented as mean ± SEM. Data were compared between groups of cells by t -testing when one comparison was performed or by ANOVA for multiple comparisons. When signifi cance was indicated by ANOVA, the Tukey-Kramer post hoc test was used to specify between-group differences. Values of P < 0.05 were considered statistically signifi cant.

Colocalization of ATP7A with macrophages in atherosclerotic lesions
In a previous report, we observed robust staining of ATP7A in murine aortic atherosclerotic lesions ( 8 ). In the tioned medium elicited much less LDL oxidation compared with that observed in the presence of cells. Moreover, a similar level of LDL oxidation was elicited by conditioned medium from ATP7A downregulated versus control cells. In contrast, cell-mediated LDL oxidation was reduced following downregulation of ATP7A. These fi ndings suggest that downregulation of ATP7A does not suppress LDL oxidation simply by preventing copper egress into the incubation medium.
Downregulation of ATP7A leads to reduced cPLA 2 ␣ expression and cPLA 2 activity Because our results suggested that downregulation of ATP7A in THP-1 macrophages inhibits cell-mediated LDL oxidation and because ablation of cPLA 2 ␣ inhibits macrophage-mediated LDL oxidation ( 19 ), we examined whether ATP7A regulates cPLA 2 ␣ expression and activity in macrophages. Figure 4 shows that expression (mRNA and protein) and activity of cPLA 2 ␣ were decreased following downregulation of ATP7A by siRNA transfection. To examine the mechanisms leading to reduced cPLA 2 ␣ expression, cells were transiently cotransfected with a plasmid encoding 2.4 kb of cPLA 2 ␣ promoter along with control or ATP7A siRNA oligonucleotides. We coupled a relatively large region of the cPLA 2 ␣ promoter upstream of the reporter gene to ensure a high likelihood that the DNA introduced contained relevant regulatory elements for transcriptional control. Downregulation of ATP7A decreased cPLA 2 ␣ promoter activity by 3-to 4-fold ( Fig.  4D ), which suggests that endogenous ATP7A transcriptionally regulates expression of cPLA 2 ␣ .
In addition to cPLA 2 ␣ , we also examined the expression of other genes reported to be involved in macrophagemediated intracellular LDL oxidation, including 12 and 15 LOX and LRP1. In contrast to the expression of cPLA 2 ␣ ( Fig. 4B ), mRNA levels of 12 LOX, 15 LOX, and LRP1 expression persisted for up to 4 d postincubation (data not shown).
Next, we investigated the role of ATP7A in macrophage mediated LDL oxidation. Figure 2B demonstrates that the THP-1-derived macrophages oxidized human LDL (250 µg/ml) over a 48 h time course, which was significantly decreased following transfection with ATP7Aspecifi c siRNA oligonucleotides compared with controls after 24, 36, or 48 h of incubation. As a negative control, LDL incubated in medium (RPMI 1640) alone (without cells) exhibited no detectable TBA reactive species (data not shown). Importantly, cell viability was similar between ATP7A-downregulated macrophages and control cells, as assessed by MTT assay (data not shown). These observations suggest that endogenous ATP7A contributes to LDL oxidation in THP-1-derived macrophages. In addition, compared with control oligonucleotides, treatment with ATP7A siRNA oligonucleotides substantially reduced O 2 • Ϫ levels (an average of 40%) in THP-1-derived macrophages (data not shown).
As ATP7A functions to egress copper from cells, and extracellular copper has potential to promote LDL oxidation, we separately examined the effects of conditioned medium versus cells on LDL oxidation. For these experiments, after downregulation of ATP7A for 48 h, the conditioned medium was harvested and incubated with native LDL for an additional 48 h. As shown in Fig. 3 , the condi-Fig. 3. Comparison of LDL oxidation mediated by cells versus conditioned medium in THP-1-derived macrophages. After incubation with ATP7A or control siRNA oligonucleotides for 48 h, the conditioned media (left two bars) from THP-1-derived macrophages were harvested and then incubated with native LDL (250 µg/ml protein) in Ham's F12 medium without FBS for an additional 48 h. Afterwards, lipid oxidation was assessed by the TBA assay, while cell mediated LDL oxidation (right two bars) was performed as described in Fig. 2B . Each data point is the mean of triplicate measurements after subtraction of cell-free control. * P < 0.01 versus ATP7A-downregulated macrophages. ns, Not signifi cant. were not affected by downregulation of ATP7A (data not shown). These results suggest that endogenous ATP7A does not indiscriminately regulate expression of enzymes implicated in macrophage-mediated oxidation of LDL.

cPLA 2 ␣ overexpression induces LDL oxidation, which is blocked by ATP7A downregulation
To further investigate whether cPLA 2 ␣ has a causative role in ATP7A-mediated LDL oxidation, cPLA 2 ␣ was overexpressed in ATP7A-downregulated THP-1 macrophages. Consistent with a previous fi nding ( 19 ) that chemical inhibitors of, and antisense oligonucleotides directed against, cPLA 2 led to decreased macrophage-mediated LDL oxidation, we found that cPLA 2 ␣ overexpression increased LDL oxidation in THP-1 cells ( Fig. 5 ). Downregulation of ATP7A abolished the increased LDL oxidation induced by overexpression of cPLA 2 ␣ . Taken together, these results suggest a role for cPLA 2 ␣ in ATP7A-mediated LDL oxidation in THP-1 cells. Fig. 5. Effects of ATP7A downregulation on LDL oxidation in cells overexpressing cPLA 2 ␣ . Cells were cotransfected with the ECGF-cPLA 2 ␣ vector or control vector along with siRNA oligonucleotides targeted to ATP7A or control. Forty-eight hours later, cells were incubated with native LDL (250 µg/ml protein) in Ham's F12 medium without FBS for additional 48 h. Afterwards, lipid oxidation was assessed by the TBA assay. Each data point is the mean of triplicate measurements after subtraction of cell-free control. * P < 0.05 versus ATP7A-downregulated or control macrophages. Fig. 4. Effect of downregulation of ATP7A on mRNA levels (A), protein levels (B), enzymatic activity (C), and promoter activity (D) of cPLA 2 ␣ in human THP-1-derived macrophages. A: Following treatment with ATP7A-specifi c or control siRNA oligonucleotides for 24 h, total RNA was isolated, and human cPLA 2 ␣ and GAPDH expression was quantifi ed by RT-PCR. The abundance of target mRNA was calculated in relation to the GAPDH mRNA in the same sample. The data are presented as fold change in mRNA expression compared with control cells (treated with control siRNA oligonucleotides). Each data point is the mean of triplicate measurements. * P < 0.05 versus ATP7A-downregulated macrophages. B and C: Following treatment with ATP7A-specifi c or control siRNA oligonucleotides for 48 h, cell lysates were prepared and the protein expression and activity of cPLA 2 ␣ was assessed as described in Fig. 2 . For Western blot, ␣ -tubulin served as a loading control. Representative blots are from three separate experiments. * P < 0.05 versus ATP7A-downregulated macrophages. D: Cells were cotransfected with the cPLA 2 ␣ -luciferase reporter plasmid (PA3-Luc/cPLA 2 ␣ ) along with control or ATP7A siRNA oligonucleotides. Forty-eight hours later, lysates were prepared, and luciferase activity (normalized to phRLTK activity) was determined as described in Materials and Methods. Results represent the mean of three independent experiments. * P < 0.01 versus ATP7A-downregulated cells.

DISCUSSION
We previously reported that ATP7A is highly expressed in the vasculature, including vascular smooth muscle cells and endothelial cells, suggesting a previously unrecognized role in cardiovascular homeostasis ( 8,33 ). Here, we report that ATP7A is robustly expressed in intimal macrophages of murine atherosclerotic aorta. Moreover, in a macrophage cell model, we demonstrate that the downregulation of ATP7A results in inhibition of LDL oxidation, at least in part through a cPLA 2 ␣ -dependent pathway.
ATP7A primarily functions to egress copper from cells, thereby supplying this cofactor to secreted coppercontaining proteins, including extracellular superoxide dismutase ( 8 ), peptidylglycine ␣ -amidating monooxygenase ( 34 ), and tyrosinase ( 35 ), through a posttranslational mechanism. These actions are facilitated by the functional domains of ATP7A, which include the NH 2 -terminal metal binding domain, the transmembrane domain, the ATPbinding domain, and the COOH-terminal di-leucine motifs. In addition, ATP7A may contain a nuclear targeting sequence ( 36 ), suggesting that it is capable of regulating gene transcription. In this regard, ATP7A-defi cient cells exhibited reduced mRNA expression of lysyl oxidase, elastin, and procollagen type I ( 37 ). Our fi nding that cPLA 2 ␣ expression, and its promoter activity, are reduced following downregulation of ATP7A in macrophages further suggests a direct role for ATP7A in regulating gene transcription. cPLA 2 ␣ , also known as group IVA PLA 2 , is the major intracellular form of PLA 2 . This phospholipase is an 85 kDa protein that harbors a Ca 2+ -binding C2 domain, two catalytic domains, a putative pleckstrin homology domain, and a number of phosphorylation sites ( 38 ). cPLA 2 ␣ preferentially hydrolyzes membrane phospholipids at the sn -2 position to release arachidonic acid, which serves as precursor for a wide spectrum of eicosanoids, including prostaglandins, thromboxanes, leukotrienes, hydroxy-and likely related to reduced expression and activity of cPLA 2 , suggesting a novel link between this phospholipase and ATP7A. These fi ndings suggest that ATP7A could play a previously unrecognized role in the pathogenesis of atherosclerosis. epoxy-fatty acids, lipoxins, and isoprostanes. cPLA 2 ␣ is activated by increased intracellular Ca 2+ concentration and by phosphorylation on multiple serine residues [see review by Leslie ( 39 )]. In addition to posttranslational control, the expression of cPLA 2 ␣ is regulated transcriptionally by a number of agents, including cytokines such as interleukin -1 ␤ ( 40 ), tumor necrosis factor-␣ , and interferon-␥ , as well as thrombin ( 41 ) and platelet-derived growth factor-BB ( 42 ). The promoter for cPLA 2 ␣ has been isolated from both humans ( 43 ) and rats ( 44 ). A number of putative binding sites for possible regulatory elements have been identifi ed within the 5 ′ -fl anking region of cPLA 2 ␣ , including AP-1 sites ( 43 ), nuclear factor B sites ( 40 ), and glucocorticoid regulatory elements ( 43 ). Our fi nding that the mRNA level of cPLA 2 ␣ is decreased after downregulation of ATP7A is further evidence of the importance of transcriptional regulation of cPLA 2 ␣ . Because both cPLA 2 ␣ and ATP7A are widely expressed in cells throughout the body, this fi nding could be of signifi cance to normal cellular physiology and to the pathology of Menkes disease.
The mechanisms whereby endogenous ATP7A contributes to LDL oxidation by THP-1 macrophages remain to be defi nitively established. We recently reported that endogenous ATP7A functions to egress copper in PMAstimulated THP-1 cells ( 9 ). As extracellular copper and other transition metals can oxidize LDL, it is conceivable that downregulation of ATP7A could modulate LDL oxidation simply by inhibiting copper effl ux into the extracellular medium. However, if such were the case, we would expect to observe reduced LDL oxidation when incubated in conditioned medium from ATP7A-downregulated cells compared with control cells. As shown in Fig. 3 , no such differences were detected. Rather, our data suggest that downregulation of ATP7A reduces oxidation of LDL by inhibiting intracellular oxidant-dependent mechanisms. To address this possibility, we examined the effects of ATP7A downregulation on the expression of enzymes implicated in macrophage-mediated oxidation of LDL. We observed that downregulation of ATP7A led to reduced expression and activity of cPLA 2 ␣ , which is thought to be involved in LDL oxidation by supplying fatty acid substrate for LOX-mediated metabolism and by inducing membrane translocation and activation of NADPH oxidase in monocytes ( 45 ). Interestingly, treatment of a murine macrophage-like cell line with PMA resulted in increased lysosomal oxidant generation, presumably due to assembly and activation of NADPH oxidase in the lysosomal membrane ( 46 ). Moreover, human monocyte-derived macrophages were recently demonstrated to oxidize LDL intracellularly through a lysosomal-dependent mechanism ( 18 ). Thus, the cellular source of oxidants, the location of LDL oxidation, and the overall importance of the ATP7A-cPLA 2 ␣ pathway in regulating LDL oxidation in macrophages remain to be determined.
In summary, we report that ATP7A is expressed in macrophages present within murine atherosclerotic lesions and that downregulation of ATP7A attenuates oxidation of LDL by THP-1-derived macrophages. The mechanism is