Oxidized Alkyl Phospholipids Are Specific, High Affinity Peroxisome Proliferator-activated Receptor gamma  Ligands and Agonists*

Sean S. DaviesDagger §, Aaron V. Pontsler||, Gopal K. Marathe||, Kathleen A. Harrison**, Robert C. Murphy**, Jerald C. HinshawDagger Dagger , Glenn D. PrestwichDagger Dagger , Andy St. Hilaire||, Stephen M. Prescott||§§, Guy A. Zimmerman||, and Thomas M. McIntyreDagger ||¶¶

From the Departments of Dagger  Pathology, || Internal Medicine, and Dagger Dagger  Medicinal Chemistry, the  Program in Human Molecular Biology and Genetics, the §§ Huntsman Cancer Institute, University of Utah, Salt Lake City, Utah 84112 and the ** Department of Pediatrics, National Jewish Medical and Research Center, Denver, Colorado 80206

Received for publication, January 30, 2001, and in revised form, February 23, 2001


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Synthetic high affinity peroxisome proliferator-activated receptor (PPAR) agonists are known, but biologic ligands are of low affinity. Oxidized low density lipoprotein (oxLDL) is inflammatory and signals through PPARs. We showed, by phospholipase A1 digestion, that PPARgamma agonists in oxLDL arise from the small pool of alkyl phosphatidylcholines in LDL. We identified an abundant oxidatively fragmented alkyl phospholipid in oxLDL, hexadecyl azelaoyl phosphatidylcholine (azPC), as a high affinity ligand and agonist for PPARgamma . [3H]azPC bound recombinant PPARgamma with an affinity (Kd(app) approx 40 nM) that was equivalent to rosiglitazone (BRL49653), and competition with rosiglitazone showed that binding occurred in the ligand-binding pocket. azPC induced PPRE reporter gene expression, as did rosiglitazone, with a half-maximal effect at 100 nM. Overexpression of PPARalpha or PPARgamma revealed that azPC was a specific PPARgamma agonist. The scavenger receptor CD36 is encoded by a PPRE-responsive gene, and azPC enhanced expression of CD36 in primary human monocytes. We found that anti-CD36 inhibited azPC uptake, and it inhibited PPRE reporter induction. Results with a small molecule phospholipid flippase mimetic suggest azPC acts intracellularly and that cellular azPC accumulation was efficient. Thus, certain alkyl phospholipid oxidation products in oxLDL are specific, high affinity extracellular ligands and agonists for PPARgamma that induce PPAR-responsive genes.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The transcription factor PPARgamma ,1 in association with its 9-cis-retinoate-binding RXR partner, controls metabolic and cellular differentiation genes that contain variations of a cognate PPAR-response element (1). PPARgamma , like other members of the broad nuclear hormone receptor family, undergoes a conformational change when it binds specific lipid ligands. This structural reorganization alters its associated proteins, releasing transcriptional inhibitors and recruiting transcriptional co-activators. The regulation of PPARgamma function is therefore controlled by lipid ligand binding.

A number of synthetic ligands for PPARgamma are known. One of these, rosiglitazone (BRL49653), binds with high affinity and is widely prescribed as an insulin sensitizer in type II diabetes. However, defining relevant biologic ligands has been problematic. Several oxidatively modified fatty acids bind and activate PPARgamma , including 15-deoxy-Delta 12,14-prostaglandin J2 (15-deoxy-PGJ2), other arachidonate metabolites (2, 3), the linoleate derivatives 9-HODE and 13-HODE (4), and several free fatty acids (5, 6). However, none of these are particularly potent agonists, and for some their presence at concentrations sufficient to activate PPARgamma can be questioned. For example, the oxygenated fatty acid products described above do not confer much advantage in potency over activation by free arachidonate (7) where a concentration of several micromolar is required to elicit a response. Moreover, the 9- and 13-HODEs and their peroxides found in oxidized LDL (8) or in skin exposed to the tumor promoter phorbol myristate acetate (9) are esterified, yet only the free forms of these lipids, and not the intact phospholipids from these sources, are PPAR ligands (10). Additionally, it is unlikely that the PPARgamma and PPARalpha agonist 15-deoxy-PGJ2 (2, 3) accumulates in vivo, and it now appears that little 15-deoxy-PGJ2 is actually present in commercial sources of this reactive and unstable lipid (11).

Oxidation of LDL creates unknown PPARgamma agonists (10). This process also creates PPARalpha ligands (12, 13), the bulk of which depend on liberation by phospholipase A2 to free fatty acid oxidation products (13). PPARalpha alters lipid metabolism, enhances lipid oxidation, and often dampens inflammatory events and signaling pathways (14, 15). PPARgamma has a distinct profile of activities as it promotes adipogenesis through differentiation of preadipocytes, and it may have a complex role in atherogenesis (1, 16). In part, its pro-atherogenic effects may occur through the formation of foam cells by stimulating CD36 expression (17, 18).

CD36 is a member of the scavenger receptor family that promotes the uptake of oxidized LDL, driving macrophages to a lipid-surfeit state characterized by foamy fatty inclusions. Cells in atherosclerotic lesions express PPARgamma (19, 20), and CD36 is induced by PPARgamma agonists present in oxidized LDL (10, 18). CD36 ligation and internalization of LDL particles oxidized by monocytes is blocked by an excess of oxidized phospholipids (21), suggesting that one or more oxidized phospholipids is a CD36 ligand responsible for the internalization of oxLDL.

Here we show that a complex lipid (i.e. a phospholipid) formed by oxidative attack on a subclass of LDL phospholipids is effectively internalized by CD36 and is a high affinity, selective PPARgamma ligand and agonist. Alkyl phosphatidylcholines, which consist of a small portion of the LDL phosphatidylcholine pool (22), are the sole precursors for these agonists because there is selectivity for the sn-1 bond in both binding and PPRE reporter activation. We conclude that certain oxidized alkyl phospholipids define a new class of high affinity agonists for PPARgamma , and because these are found in oxidized LDL, they may contribute to its biologic effects.

    EXPERIMENTAL PROCEDURES
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Materials-- PAF was from Avanti Polar Lipids; lyso-PAF (1-O-hexadecyl-sn-glycero-3-phosphocholine), 9-cis-retinoate, pirinixic acid (WY14643), and 15-deoxy-PGJ2 were from Biomol; Rhizopus arrhizus lipase was from Roche Molecular Biochemicals and then Sigma; [3H]rosiglitazone and rosiglitazone were from the American Radiolabeled Chemicals. ECL kits were from Amersham Pharmacia Biotech; the SV40-beta -galactosidase reporter was from Promega (Madison, WI); the CD36 reporter constructs with (-273) and without (-261) its PPRE (17) were constructed from its reported sequence (23). The forward primers (for CD36-273) were 5'-GCGACGCGTCTGGCCTCTGACTTACTTGG-3' or (CD36-261) 5'-GCGACGCGTTTACTTGGATGGGAACTAGCC-3', and the reverse primer was 5'-GGAAGATCTAGTCCTACACTGCAGTCCTC-3'. The amplicon was inserted into pGL3b at the MluI and BcgII sites. pGL3b was from Promega, and Probond Ni+ beads were from Invitrogen. The blocking anti-CD36 antibody 185-1G2, without azide, was from NeoMarkers (Fremont, CA); the FITC-conjugated anti-CD36 antibody CLB-IVC7 used for flow analysis was from Accurate Chemicals (Westbury, NY); and the anti-ICAM-3 antibody CAL3.10 (BBA29) was from R & D Systems (Minneapolis MN). The flippase mimetic I (24) tris (tosylaminoethyl)amine (TTA) was synthesized as described (25).

Oxidation and Analysis of LDL and Synthetic Phospholipids-- LDL was oxidized with 20 µM CuSO4 at 37 oC overnight, and oxidized phospholipids were purified by RP-HPLC (26). A portion of the recovered fractions was treated with phospholipase A1 as before (27), except that the enzyme was from Sigma. The lipid azPC was synthesized from 1-O-hexadecyl-sn-glycero-3-phosphocholine (hexadecyl lyso-PC; after mild alkaline hydrolysis; 0.5 N NaOH in methanol; 4 h; 24 oC) and 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (palmitoyl azelaoyl-PC) was synthesized in a similar fashion from palmitoyl lyso-PC. After neutralization, purified lipid (2 mg) was reacted with 10 mg of azelaic anhydride (University of Utah Chemical Synthesis Facility) in the presence of 1 mg of 4-(N,N-dimethylamino)pyridine in CHCl3:pyridine (4:1) for 36 h before purification by RP-HPLC. The mass of each synthetic phospholipid was determined by phosphorus analysis (28). Mass spectroscopy of lipid oxidation products was performed as before (27). [3H]Hexadecyl azelaic phosphatidylcholine was synthesized from [3H]hexadecyl-sn-glycero-3-phosphocholine (PerkinElmer Life Sciences) and HPLC-purified in a similar fashion.

Cell Preparation-- Human monocytes were isolated by counter-current elutriation (29) and resuspended (1 × 106/ml) in Hanks' balanced salt solution with 0.5% human serum albumin and 10 µg/ml polymyxin B. Monocytes were added to plates coated with 10 µg/ml CAL3.10 anti-ICAM-3 monoclonal antibody (30). CV-1 cells were obtained from ATCC and grown as suggested. Surface expression of CD36 on primary human monocytes was determined by allowing elutriated monocytes to adhere to anti-ICAM3-coated wells for 1 h before the cells were exposed to the lipid agonists, or not, as stated in the figures. Some cells were maintained in a suspended state by gently rocking on a platform rocker in polypropylene tubes as a control. Adherent cells were released from the plate by gentle agitation and scraping and washed three times in PBS containing 1% goat serum. Recovered cells were stained with FITC-labeled anti-CD36 antibody CLB-IVC7 for flow analysis by the University of Utah flow analysis core facility.

Plasmids-- The acyl-CoA oxidase-luciferase plasmid was described previously (31). Plasmids were transformed into TOP10F' Escherichia coli strain using the TA cloning kit. Plasmids from log phase cells were isolated using a Bigger Prep kit (5 Prime right-arrow 3 Prime, Inc., Boulder, CO), and purified by CsCl gradients. The His6-tagged PPARgamma was constructed similarly using the M13 primer in pCR2.1 that contained full-length PPARgamma 1 and a primer (5'-CTA ATG ATG ATG ATG ATG ATG GTA CAA GTC CTT GTA G-3') containing the His6 tag sequence. PPARgamma and PPARalpha expression plasmids were a gift from Beth Meade (University of Utah). Inserts in all plasmids were sequence-verified by the University of Utah sequencing core facility.

Transfection of Cultured Cells and Reporter Assays-- When PPAR expression plasmids were co-transfected with a reporter construct, 0.5 µg of the relevant plasmid was combined with 1 µg of pGL3-PPRE and 0.1 µg of the SV40-beta -galactosidase reporter to normalize transfection efficiencies. All transfections included 1-2 µg of total plasmid, and 5-10 µl of LipofectAMINE per ml of Opti-MEM. Transfection solution was added to CV-1 cells overnight and then removed, and agonist was added in fresh media for 18-20 h. [3H]Rosiglitazone displacement from PPARgamma was determined with a carboxy His6-tagged molecule. HeLa cells were transfected with pCR3.1-PPARgamma -His6 or pCR3.1 for 21 h with LipofectAMINE and then grown (48 h). The cells were washed twice with PBS and lysed in PBS containing 0.1% Triton X-100 and frozen at -70 °C until used. Transfection with PPARgamma -His6 was assessed by immunoblotting with anti-His6 antibody (Santa Cruz Biotechnology). Debris was removed from thawed samples, and 200 µl of lysate was incubated with 50 µl of Probond beads at 4 °C for 1 h in PBS. The beads were washed once by centrifugation before [3H]rosiglitazone, and then unlabeled competitor was added in a final volume of 300 µl of PBS. Samples were incubated with shaking for 3 h at 4 °C before washing three times with PBS and quantitating retained 3H. Binding of [3H]azelaic phosphatidylcholine to PPARgamma was performed in a similar fashion, although the amount of protein lysate was increased to account for its lower specific radioactivity. Accumulation of [3H]azPC was estimated by incubating monolayers with carrier-free [3H]azPC (22.9 Ci/mmol) for 30 min in the presence or absence of the lipids specified in the figure at a concentration of 10 µM (except HPLC fraction 6 from oxLDL that was used at a concentration that maximally induced ACox reporter expression since there was insufficient material for phosphorus quantitation). Some cells were preincubated for 30 min with 10 µg of the blocking anti-CD36 antibody 185-1G2 or an irrelevant IgG2a isotype-matched control monoclonal antibody.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Oxidized Alkyl Phosphatidylcholines in Oxidized LDL Stimulate Luciferase Expression Under the Control of the Acyl-CoA Oxidase PPRE-- We oxidized human LDL, extracted the lipids, and separated nonpolar lipids (which we found to have no activity in this assay; not shown) from the polar phospholipid oxidation products. We separately examined these polar phospholipid-containing fractions as agonists using CV-1 cells that had been transiently transfected with an acyl-CoA oxidase PPRE-firefly luciferase reporter construct and SV40-beta -galactosidase to normalize for transfection efficiency. These purified polar phospholipids stimulated luciferase transcription under the control of this PPRE (Fig. 1), and this activity was concentrated in RP-HPLC fraction 6. We found this material to be as effective an agonist for PPAR-induced transcription as rosiglitazone (BRL49653), and a synthetic oxidized phospholipid (azPC) is discussed in detail below. Fig. 1 shows that an equivalent fraction from the same batch of LDL that had not been oxidized contained little stimulatory activity.


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Fig. 1.   oxLDL contains PPRE agonists that are resistant to phospholipase A1 digestion. CV-1 cells were transiently transfected with a luciferase reporter under the control of the PPRE from acyl-CoA oxidase and an SV40-beta -galactosidase plasmid to normalize transfection efficiency. These cells were then treated for 18 h with buffer, 1 µM rosiglitazone (Rosi) (BRL49653), 1 µM of the synthetic oxidized phospholipid azPC, or equivalent volumes of HPLC fractions of lipids extracted from native or oxidized (Ox) human LDL. Each reporter was then assayed as described under "Experimental Procedures" and their ratio calculated. Polar phospholipids contained in Cu+-oxidized LDL were purified by RP-HPLC, and the fractions eluting at min 6-8 were collected; the solvent was removed by a stream of N2; and the remaining lipids were resuspended in Hanks' balanced salt solution/A by a brief sonication. Polar phospholipid oxidation products with PAF-like activity elute at min 5 and 6 and PAF elutes in fraction 7 (27) in this system. An aliquot of the polar phospholipids isolated from oxidized LDL was treated with phospholipase A1 and re-extracted prior to inclusion in the assay. Data are presented as the range of two determinations and represent results of two separate experiments.

Oxidation of LDL creates oxidized phosphatidylcholines that are potent inflammatory agents because they structurally resemble PAF and activate the cloned receptor for PAF. Oxidized phospholipids of this class are all derived from the small pool of alkyl phosphatidylcholines in LDL (22) that are resistant to phospholipase A1 digestion (27). To determine whether the oxidatively generated agonists that induce transcription from a PPRE reporter construct also fall into this class, we digested the fractions isolated by HPLC with phospholipase A1. This removes the oxidized diacyl phospholipids, derived from the 99.5% of LDL phospholipids that have an sn-1 ester bond, as assessed by phosphorus analysis (not shown). Phospholipase A1 digestion had no effect on the ability of the fractions isolated from oxidized LDL to stimulate luciferase expression from the PPRE-reporter construct (Fig. 1), indicating that only alkyl phosphatidylcholine oxidation products were effective agonists in this assay.

Azelaoyl Phosphatidylcholine Is a Prominent Oxidation Product in Oxidized LDL-- We determined which alkyl phosphatidylcholine oxidation products were present in oxidized LDL by resolving the phospholipase A1-treated phospholipids by reversed phase HPLC and examining these by electrospray tandem mass spectroscopy as precursors of the phosphocholine ion m/z 184. An abundant ion in the HPLC effluent was observed at m/z 652 (Fig. 2a), potentially corresponding to 1-O-hexadecyl-2-azelaoyl-sn-glycero-3-phosphocholine (azPC). This component was maximal in RP-HPLC fraction 6 (Fig. 2, inset), whereas the C4-PAF analogs were most abundant in fractions 7 and 8, as reported previously (27). A structurally unique diagnostic product of sn-2 omega -carboxyl glycerophosphocholine lipids is the collision-induced rearrangement of a methyl group and decomposition to a monomethyl acid and dimethyl lyso-PC (32). Proof that azPC was present was obtained by collision-induced decomposition of the corresponding [M - H]- of m/z 650 (Fig. 2b) that yielded the expected product ions at m/z 201 and 466 corresponding to monomethyl azelaic acid and the dimethyl lyso-PAF adduct resulting from the loss of the sn-2 methylazelaoyl ketene. azPC is therefore an abundant oxidation product of LDL alkyl phosphatidylcholines. We confirmed this deduction by synthesizing azPC and finding that synthetic azPC produced the same fragmentation pattern as the material isolated from oxidized LDL (not shown).


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Fig. 2.   Identification of hexadecyl azelaoyl phosphatidylcholine in oxidized LDL. a, electrospray tandem mass spectroscopy of precursor-positive ions that yield m/z 184 ions (phosphocholine cation) in oxidized LDL. LDL was oxidized, the phospholipid fraction collected by aminopropyl chromatography, and treated with phospholipase A1. The unhydrolyzed alkyl polar phospholipids were re-extracted, resolved by RP-HPLC, and collected as they eluted from the column as in Fig. 1. Mass spectral analysis of a portion of each fraction revealed the population of [M + H]+ ions derived from phosphatidylcholine or sphingomyelin generated by electrospray ionization included m/z 652, the expected [M + H]+ for azPC, as a predominant component of RP-HPLC fraction 6. Inset, abundance of [M + H]- ions derived from azPC (m/z 652) and C4-PAF (m/z 552) during flow injection analysis of fractions collected during RP-HPLC separation of oxidized LDL phospholipids. b, product ions obtained following collision-induced decomposition of the corresponding negative ion [M - H]- at m/z 650 revealed the diagnostic carboxylate anion m/z 201 corresponding to the monomethyl ester of azelaic acid and the dimethyl lyso-PAF anion at m/z 466 formed by loss of the sn-2 substituent as a ketene.

Synthetic Hexadecyl Azelaoyl Phosphatidylcholine Is a High Affinity Ligand for PPARgamma -- We experimentally determined whether a complex lipid like azPC could function as a ligand for PPARgamma . We synthesized [3H]hexadecyl azelaoyl phosphatidylcholine ([3H]azPC) and incubated it with full-length recombinant human PPARgamma 1. The PPARgamma in transfected HeLa cell lysates was immobilized on Ni+ beads through an introduced His6 tag, so tight binding could be assessed after collecting and washing the beads. We found (Fig. 3a) that [3H]azPC bound to PPARgamma , and that this binding was dependent on the concentration of immobilized hPPARgamma 1. We next varied the concentration of [3H]azPC to establish its apparent affinity for PPARgamma under these conditions. We found that the binding of [3H]azPC was concentration-dependent and that its apparent affinity was approx 40 nM (Fig. 3b). However, we also found that different lysates provided different apparent affinities, perhaps reflecting a similar wide disparity in reported apparent affinities for rosigitazone (2, 33, 34). To determine better whether azPC bound PPARgamma as effectively as rosigitazone, we directly compared [3H]azPC binding with [3H]rosiglitazone binding at low concentrations and found (Fig. 3c) that [3H]azPC binding precisely mirrored the binding of [3H]rosiglitazone to immobilized PPARgamma 1.


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Fig. 3.   azPC is a high affinity ligand for PPARgamma . a, [3H]azPC binds to immobilized full-length PPARgamma . Lysates were prepared from HeLa cells transiently transfected with plasmid pCR3.1-PPARgamma -His6 or pCR3.1, and the ectopic protein from the stated amount of lysate was immobilized on Ni+ beads. Charged or uncharged beads were incubated with [3H]azPC before the beads were recovered and washed, and associated radioactivity was quantitated by liquid scintillation counting. b, [3H]azPC binding to immobilized PPARgamma as a function of azPC concentration. hPPARgamma 1-His6 immobilized on Ni+ beads was incubated with the stated concentration of azPC before bound material was quantitated as above. The range of duplicate points from 1 of 3 representative experiments is presented. c, direct comparison of [3H]azPC and [3H]rosiglitazone binding at low concentrations. The amount of carrier-free [3H]azPC or [3H]rosiglitazone, whose concentrations were calculated from their specific radioactivity, retained by immobilized PPARgamma 1-His6 was determined as in the above experiments.

Synthetic azPC Interacts with the Ligand-binding Pocket of PPARgamma -- Rosiglitazone co-crystallizes with the ligand-binding domain of PPARgamma (35, 36) in the ligand-binding pocket, so displacement of [3H]rosiglitazone tests binding in this pocket. We found (Fig. 4a) that azPC displaced the standard ligand [3H]rosiglitazone in a concentration-dependent fashion and that the concentration relationship of this competition was identical to that of unlabeled rosiglitazone. azPC bound PPARgamma only through this ligand-binding pocket because this competition with rosiglitazone was complete. We examined the effect of other lipids as competitors at 6.7 µM where azPC and rosiglitazone competition was complete and found (Fig. 4b) that PAF and 15-deoxy-PGJ2 could only displaced about a third of the [3H]rosiglitazone at this concentration. Free azelaic acid, like arachidonate, was an ineffective competitor.


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Fig. 4.   azPC competes for rosiglitazone (Rosi) binding in the PPARgamma ligand-binding pocket. a, azPC displacement of [3H]rosiglitazone from PPARgamma . [3H]Rosiglitazone (0.33 nM) was mixed with the stated concentration of unlabeled rosiglitazone or azPC before the amount of [3H]rosiglitazone retained by immobilized PPARgamma was determined. An excess (13.6 µM) of unlabeled rosiglitazone in these transfected Chinese hamster ovary cell lysates reduced binding to the level of untransfected cells. b, azPC is a preferred PPARgamma ligand. Competitive displacement of [3H]rosiglitazone from Ni+-bound PPARgamma was determined with an excess (6.7 µM) of the stated compound. c, azPC binds to immobilized PPARgamma and is equally displaced by rosiglitazone or unlabeled azPC. The stated concentration of azPC or rosiglitazone was incubated with His6-PPARgamma that had been mixed with [3H]azPC before the amount of retained radiolabel was determined as above. d, lipid displacement of azPC is selective. Interference of [3H]azPC binding to PPARgamma was analyzed as in b with an excess (6.7 µM) of the stated lipid. Neg, negative.

We performed the converse experiment where unlabeled rosiglitazone or azPC was used to displace [3H]azPC bound to immobilized PPARgamma . We found (Fig. 4c) that unlabeled rosiglitazone displaced nearly all of the [3H]azPC from PPARgamma as its concentration was increased, just as unlabeled azPC displaced this bound [3H]azPC. We tested other lipids for their ability to displace [3H]azPC and found (Fig. 4d) that PAF, and to a lesser extent lyso-PAF, was a modest competitor, but that 9-HODE or 13-HODE were unable to displace bound [3H]azPC. We tested palmitoyl azelaoyl-PC, the diacyl homolog of hexadecyl azelaoyl PC, as a PPARgamma ligand, and we found it to be 10-100-fold less potent as a competitor (not shown).

azPC Is a Potent Agonist for PPAR-responsive Elements-- azPC was a ligand for PPARgamma , so we next determined whether it was an agonist. We transfected CV-1 cells with a luciferase reporter under the control of the PPRE from acyl-CoA oxidase, along with SV40-beta -galactosidase as a transfection control, and then treated the cells with rosiglitazone, synthetic azPC, or 9-cis-retinoate to activate RXR. Rosiglitazone induced a 3.4-fold increase in reporter expression, and azPC induced a 3.9-fold increase (Fig. 5a) in this assay. Fig. 1 presented a similar result where azPC induced a 3.9-fold increase in ACox reporter expression. Activation of just the RXR subunit with 9-cis-retinoate, which can act as a phantom ligand (37), was not as effective as either PPARgamma ligand in stimulating reporter expression.


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Fig. 5.   a, azPC fully stimulates reporter expression controlled by acyl-CoA oxidase PPRE. CV-1 cells were transfected with the acyl-CoA-oxidase-PPRE-luciferase (ACox) reporter construct along with SV40-beta -galactosidase to normalize transfection efficiency. After 1 h of recovery, these cells were treated for 18 h with 200 nM rosiglitazone (Rosi), 200 nM azPC, or 1 µM 9-cis-retinoate (RA). Firefly luciferase and SV40-beta -galactosidase activities in the cellular lysates were determined, and the data are presented as this normalized ratio. b, azPC stimulation of normalized ACox-luciferase expression is concentration-dependent. CV-1 cells were transfected with ACox-PPRE-luciferase and SV40-beta -galactosidase and then stimulated for 24 h with buffer, or the stated amount of synthetic azPC, diacyl azPC, or rosiglitazone. These are representative results from one of two experiments.

We compared the concentration-response relationship of azPC and its acyl analog with rosiglitazone as PPARgamma agonists. CV-1 cells were transfected with the ACox reporter and SV40-beta -galactosidase for normalization, and we then treated with increasing concentrations of azPC, diacyl azPC, or rosiglitazone. We found that azPC induced a concentration-dependent increase in reporter expression starting by about 10-8 M (Fig. 5b). Half-maximal activation occurred by 10-7 M, and maximal expression was achieved by 1 µM. Rosiglitazone also induced a concentration-dependent increase, and this relationship was identical to that of azPC. We also examined the diacyl homolog of azPC, 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine, as an agonist; we found that it was about 100-fold less effective than azPC and only began to elicit an effect at 10 µM.

azPC Is a Specific PPARgamma Agonist-- We determined the specificity of azPC as a PPARgamma agonist by transfecting CV-1 cells with PPARalpha or PPARgamma expression plasmids in addition to the ACox reporter and an SV40 beta -galactosidase transfection control. Rosiglitazone induced an 8-fold increase in reporter expression in this experiment (Fig. 6) in untransfected cells that rely on their endogenous PPARs. This was a more robust response than the 5.3-fold increase induced by the PPARalpha -selective agonist WY14643 or the 5-fold increase induced by azPC in these control cells. When PPARgamma was overexpressed in these cells, the response to rosiglitazone was markedly enhanced (a 25-fold induction), as was the response to azPC (an 18-fold induction). In contrast, overexpression of PPARalpha failed to enhance the existing response to either azPC or rosiglitazone. The ectopic PPARalpha was functional because the PPARalpha -selective agonist WY14643 enhanced expression in cells overexpressing PPARalpha and not in cells transfected with PPARgamma .


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Fig. 6.   azPC is a selective PPARgamma agonist. CV-1 cells were transfected with acyl-CoA oxidase-PPRE-luciferase and SV40-beta -galactosidase without or with co-transfection with PPARalpha or PPARgamma expression plasmids. These cells were then exposed to buffer, 1 µM rosiglitazone (Rosi), or azPC, or 5 µM WY14643 for 24 h before the ratio of luciferase to beta -galactosidase was determined as before. These results are representative of two separate experiments.

CD36 Is Induced by azPC through Its PPRE-- We determined whether endogenous PPAR-regulated genes were induced by azPC, and for this we chose CD36, a scavenger receptor that binds and internalizes oxidized LDL particles. Intriguingly, transcription of CD36 in monocytes is stimulated by unknown ligands associated with oxidized LDL (10, 18). We exposed adherent primary human monocytes to rosiglitazone, and we found that it increased the surface expression of CD36 as expected (Fig. 7a). There was an equivalent enhancement of CD36 surface expression when monocytes were exposed to synthetic azPC. Additionally, we found that HPLC fraction 6 of oxidized LDL was just as effective as azPC and rosiglitazone in enhancing CD36 surface expression. Finally, we found that just adhesion and spreading of the monocytes in the absence of one of these ligands had no effect on surface CD36 expression.


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Fig. 7.   CD36 is induced by azPC through its PPRE. a, azPC enhances CD36 surface expression in primary human monocytes. Monocytes freshly isolated by elutriation were allowed to adhere to anti-ICAM3-coated wells and then incubated with buffer, 1 µM azPC, rosiglitazone (Rosi), or with fraction 6 of HPLC-purified polar phospholipids derived from oxidized LDL. After overnight incubation, these cells were released from the plate and stained with FITC-labeled anti-CD36 antibody CLB-IVC7. The upper left panel shows that adhesion alone was not stimulatory as CD36 expression by unactivated cells held in suspension was the same as those allowed to adhere to anti-ICAM3-coated surfaces. b, azPC stimulates CD36 transcription through a PPRE and PPARgamma . CV-1 cells were transfected with a CD36 promoter-luciferase reporter that contained its PPRE (CD36-273) or one that did not (CD36-261). All cells were co-transfected with SV40-beta -galactosidase, and some cells were additionally transfected with expression plasmids encoding RXRalpha , PPARalpha , or PPARgamma . The cells were then treated for 16 h with 1 µM rosiglitazone or azPC before the ratio of luciferase to beta -galactosidase was determined and normalized to buffer-treated cells. These results are representative of a separate experiment.

We determined whether azPC stimulated CD36 transcription by using CD36 promoter-reporter constructs that either contained its PPRE at position -273 (pCD36-273) or lacked this element (pCD36-261). We found (Fig. 7b) that rosiglitazone enhanced pCD36-273 reporter expression in CV-1 cells through endogenous receptors by nearly 3-fold and that exposure to azPC produced an identical response. This induction did not occur when we used pCD36-261 that lacked the PPRE. We next co-transfected these cells with RXR, PPARalpha , or both RXR and PPARalpha expression plasmids, and we found that forced expression of these nuclear hormone receptors did not substantially alter these results. However, when the cells were co-transfected with PPARgamma , either alone or in combination with RXR, the induction of pCD36-273 by rosiglitazone was markedly enhanced. The response of the pCD36-273 reporter plasmid to azPC was equally augmented by overexpression of PPARgamma . This augmentation only occurred when the reporter construct contained the PPAR-responsive element, so activation of PPARgamma , and not PPARalpha , by azPC stimulates CD36 expression via this PPRE.

CD36 Aids in the Uptake of Extracellular azPC-- CD36 translocates entire oxidized lipoprotein particles into cells, apparently by binding to incorporated oxidized phospholipids (21). We determined whether CD36 also transports extracellular oxidized phospholipids not incorporated into a lipoprotein particle. We found that human monocytes accumulated [3H]azPC and that this accumulation was reduced by excess unlabeled azPC or the polar phospholipids isolated from oxidized LDL (Fig. 8a). Excess PAF was less effective in blocking [3H]azPC accumulation and inhibited about half of the [3H]azPC accumulation. The blocking anti-CD36 antibody 185-1G2 also blocked half of the specific accumulation of [3H]azPC, whereas an isotype-matched control monoclonal antibody had no effect. We next transfected CV-1 cells with the ACox PPRE reporter plasmid and then treated the cells with the blocking anti-CD36 antibody prior to exposing these cells to azPC or the stimulatory polar phospholipids purified from oxidized LDL. We found (Fig. 8b) that the inhibitory monoclonal antibody 185-1G2 effectively blocked the induction of the reporter in response to either azPC or the polar phospholipids isolated from oxidized LDL. This effect did not extend to all lipids as luciferase expression in response to rosiglitazone was not inhibited by the monoclonal antibody.


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Fig. 8.   CD36 aids azPC accumulation and induction of ACox PPRE-driven transcription. a, [3H]azPC accumulation is blocked by anti-CD36 antibody. Monocytes were incubated with [3H]azPC for 30 min without or with the addition of the stated lipids at a concentration of 10 µM. Some cells were preincubated with 10 µg/ml blocking 185-1G2 anti-CD36 antibody or an isotype-matched IgG2a control antibody. b, inhibition of CD36 function inhibits azPC-induced PPRE-controlled luciferase expression. CV-1 cells were transfected with ACox-luciferase and SV40-beta -galactosidase as above and then treated with the CD36 blocking antibody 185-1G2 prior to addition of 1 µM rosiglitazone (Rosi), azPC, or intact oxidized LDL particles. The fold induction of the normalized luciferase to beta -galactosidase was determined as before, and this experiment represents the results of an independent experiment.

Extracellular azPC Has Ready Access to Intracellular PPARgamma -- Our results suggest that azPC is a direct agonist for PPARgamma , but it might be argued that azPC activates a surface receptor whose signal induces the synthesis of the true intracellular ligand for this nuclear receptor. In this case, enhancing intracellular access of azPC should have no effect on reporter induction. We tested this postulate with the small molecule flippase mimetic TTA (Fig. 9a) that facilitates anionic phospholipid flip-flop (24). We found that TTA had no effect of its own on ACox reporter expression in CV-1 cells (not shown) but that it doubled the 1.7-fold induction of the acyl-CoA oxidase reporter induced by azPC alone (Fig. 9b). This enhancement in azPC activity by TTA was concentration-dependent with little sign of toxicity up to 37 µM, the concentration used to study vesicular transport (24). This cationic lipid also doubled reporter expression when the extracellular agonist was an intact oxidized LDL particle (Fig. 9c), suggesting that it can exchange agonists from these particles as well. This is not the anticipated result if gene induction depended on signaling from an externally disposed plasma membrane receptor for azPC. We conclude from this, first that the flippase mimetic aids azPC penetration into cells and thereby enhances transcription by nuclear PPARgamma . Second, we conclude that because the enhancement was quite modest that azPC transport already occurs at a rate that supplies near-maximal amounts of extracellular azPC, or oxidized ligands in oxidized LDL particles, to intracellular PPARgamma .


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Fig. 9.   The flippase mimetic TTA modestly improves azPC induction of PPRE reporter expression. a, TTA complexed with azPC. The proposed interaction of TTA and azPC is based on the proposed interaction with lipid phosphodiesters (24, 25). TTA transports anionic phospholipids across membrane bilayers (24) and should circumvent cellular transport mechanisms. b, TTA affects azPC-induced gene expression. CV-1 cells were transfected with the ACox-luciferase reporter and stimulated with 200 nM azPC as described in Fig. 5. This fixed amount of azPC was preincubated with the stated concentration of the flippase mimetic I (TTA), and its effect on activation of the ACox-luciferase reporter was then determined as above. c, TTA improves azPC and intact oxidized LDL induction of ACox-luciferase expression. CV-1 cells were transfected with the ACox-luciferase reporter and treated with 200 nM azPC or an equivalent amount of oxidized LDL with or without the addition of 10 µM TTA. Luciferase was assayed as before, and an independent experiment confirmed these findings.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Our findings show that oxidatively modified phosphatidylcholines from oxidized LDL are high affinity ligands and agonists for PPARgamma . We find that there is a distinct selectivity for an sn-1 ether bond in both binding to PPARgamma and its activation by oxidized phospholipids. The selective recognition of the sn-1 ether bond means that the LDL precursors of these high affinity ligands are derived from the half percent or so of alkyl phosphatidylcholines in the pool of LDL phosphatidylcholine (22). One prominent oxidation product of this subclass was as potent as the synthetic high affinity ligand rosiglitazone.

PPARgamma is a ligand-dependent transcriptional activator where the known ligands are lipids with a remarkably wide range of structures. Crystallography reveals a large hydrophobic ligand cavity that is only 25-40% occupied by rosiglitazone (35, 36), suggesting that a variety of structures might be able to be accommodated by this pocket. azPC bound PPARgamma in a way that was saturable and was completely displaced by rosiglitazone. This latter attribute strongly suggests that azPC binds in the ligand-binding pocket.

We did not established the Kd for azPC through a Scatchard analysis, because we do not have sufficient immobilized protein for quantitation, but rather have established its apparent binding constant. The value we obtained for an apparent affinity of azPC for PPARgamma was around 40 nM, but we also found variation in this number using lysates from other transfections. More relevant was our observation that in a direct comparison there was no discernible difference between [3H]rosiglitazone and [3H]azPC binding. The affinity of rosiglitazone itself for PPARgamma is subject to variability in the literature ranging from 40 to 60 nM for the displacement of [3H]SB-236636 (38) to 200 nM for the displacement of [3H]rosiglitazone (39). These competitive displacement values span the range of values reported for the actual binding constant for rosiglitazone to PPARgamma , where the Kd ranges from 40 (33) to 100 (34) to 325 nM (2).

azPC binding to PPARgamma was a property of the whole anionic phospholipid structure, because free and unesterified azelaic acid did not compete for rosiglitazone binding. However, not all polar alkyl phosphatidylcholines interact well with PPARgamma because PAF was a poor ligand that failed to induce PPRE reporter expression. Nevertheless the sn-1 ether bond is an important structural determinant because the diacyl homolog of azPC was a poor ligand (not shown) and an even less effective agonist that only began to induce PPRE reporter function by 10 µM. This selectivity for the sn-1 ether bond is the basis for our observation that phospholipase A1 treatment of the mixed oxidized phospholipid products generated by LDL oxidation did not detectably reduce PPRE reporter activity.

The diacyl azPC analog will be formed in parallel with alkyl azPC during LDL oxidation (40) and in about 200-fold greater abundance (22). However, the results with phospholipase A1 show this numerical advantage still is not sufficient to contribute to PPARgamma activation by oxidized LDL. When considering the importance of the sn-1 bond, it is important to note that commercial preparations of lysophosphatidylcholines, used in the preparation of diacyl phosphatidylcholines with specific sn-2 residues, are variably contaminated with alkyl species.2 This can contribute to the apparent activation of PPARgamma by oxidized synthetic diacyl phosphatidylcholines, so the material we used to synthesize the diacyl homolog of azPC was first purified to avoid this spurious effect.

Oxidation of LDL creates phosphatidylcholines with fragmented sn-2 residues, some with an omega -carboxylate function (40, 41). These oxidation products are found in human plasma (42) and atherosclerotic lesions (43). PPARgamma , in contrast to PPARalpha , is aberrantly expressed in atherosclerotic lesions (20) and colonic tumors (44), which provide phospholipid oxidation products with the potential to alter the complement of genes expressed in such areas. Certain synthetic diacyl phospholipid oxidation products modestly activate PPARalpha function (12). However, PPARalpha activation by oxidized phospholipids depends on phospholipase A2 activity (13), suggesting that the oxidized free fatty acid products of this reaction are the actual ligands for PPARalpha . By contrast, we show PPARgamma to directly bind intact, and only intact, oxidized phospholipid.

The ability of azPC to drive PPRE-reporter constructs at submicromolar concentrations shows that it readily crosses cellular membranes, apparently with minimal metabolism, to selectively activate this nuclear receptor. Uptake of the intact phospholipid was a property of CD36. CD36 is a type B scavenger receptor that can account for up to half the binding, internalization, and degradation of oxidized LDL by human macrophages (45). Expression of this receptor is induced by PPARgamma agonists in oxidized LDL (10, 18). CD36 recognizes the lipid portion of oxidized LDL (45), and diacyl-oxidized phospholipids interfere with this uptake (21), suggesting that CD36 binds phosphatidylcholine oxidation products. Here we show that it also internalized at least one of them, and we find that azPC induction of nuclear transcription from the PPRE reporter was CD36-dependent. We conclude that the synthetic oxidized phosphatidylcholine was transported as an intact molecule because any hydrolysis and resynthesis likely would have generated the inactive diacyl species from cellular lysophosphatidylcholine.

We determined whether azPC uptake was limiting using TTA, a lipophilic tridentate phosphate chelator (25) that increases the rate of anionic phospholipid flip-flop across unilamellar vesicles (24). Our experiments represent the first use of this class of agents in living cells, and we found TTA to double reporter expression by azPC or intact oxidized LDL. There were two notable results here. One was that the effectiveness of azPC was enhanced by the flippase mimetic. This suggests that azPC, and lipids in oxidized LDL, directly activates PPARgamma rather than some secondary intracellular messenger produced after an initial interaction of azPC with an unknown surface receptor. The second key observation was that TTA only doubled reporter activity. If cellular uptake of exogenous azPC had been severely limiting, then circumventing this step should have markedly enhanced reporter induction. Since it did not, it is fair to conclude that azPC entry into cells is relatively efficient.

Oxidative stress arises from a number of sources, from exuberant inflammatory reactions to ionizing radiation. Reactive oxygen species and the oxidized LDL generated by them are postulated to initiate and maintain an inflammatory state in the vascular wall during atherogenesis (46-48). LDL oxidized ex vivo, like the oxidized particles obtained from atherosclerotic plaques (49), contains inflammatory lipids that increase the atherogenicity of these particles. Similarly, oxidized LDL in the circulation (42, 50) or in atherosclerotic plaques (43) also contains fragmented and oxidatively modified phospholipids (26, 40, 43). Some of these are agonists for the PAF receptor (27, 51). Here we show that others are high affinity ligands and activators of PPARgamma . This establishes a new link connecting LDL oxidation with the induction of PPAR-regulated genes, and oxidized LDL (52), fragmented phospholipids (43), monocytes (53), and PPARgamma (20) are all present in atherosclerotic lesions.

    ACKNOWLEDGEMENTS

We thank Elizabeth Meade for supplying the acyl-CoA oxidase-luciferase reporter, pCR2.1-PPARgamma , and pCR2.1-PPARalpha . We thank Clare Amann and Anna Pavlovic of the University of Utah Chemical Synthesis Facility for the synthesis and analysis of azelaic anhydride and Andrew Maxfield for aid with the synthesis of TTA. We also thank Diana Lim for figure preparation. The University of Utah DNA Synthesis and Analysis core facility and the Flow cytometry core facility were supported in part by funds from National Institutes of Health Grant P30 CA 42014.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants HL44513, HL 35217, HL34303, NS29632, and HL 44525, the Utah Centers of Excellence Program, and the Margolis Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Current address: Division of Clinical Pharmacology, Vanderbilt University Medical Center, Nashville, TN 37232.

§§ H. A. and Edna Benning Professor of Human Molecular Biology.

¶¶ To whom correspondence should be addressed: 4130 EIHG, 15 North 2030 East, University of Utah, Salt Lake City, UT 84112-5330. Tel.: 801-585-0716; Fax: 801-585-0701; E-mail: tom.mcintyre@hmbg.utah.edu.

Published, JBC Papers in Press, February 26, 2001, DOI 10.1074/jbc.M100878200

2 G. K. Marathe A. Silva, H. C. C. F. Neto, L. W. T. Joelker, S. M. Prescott, G. A. Zimmerman, and T. M. McIntyre, submitted for publication.

    ABBREVIATIONS

The abbreviations used are: PPAR, peroxisome proliferator-activated receptor; azPC, 1-hexadecyl-2-azelaoyl-sn-glycero-3-phosphocholine; TTA, tris tosylamine; LDL, low density lipoprotein; oxLDL, oxidized LDL; PPRE, PPAR-response element; RXR, retinoid X receptor; HODE, hydroxyoctadecadienoic acid; PG, prostaglandin; 15-deoxy-PGJ2, 15-deoxy-Delta 12,14-prostaglandin J2; HPLC, high pressure liquid chromatography; RP-HPLC, reversed phase HPLC; PBS, phosphate-buffered saline; PAF, platelet-activating factor; PC, phosphatidylcholine; FITC, fluorescein isothiocyanate.

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DISCUSSION
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