From the Departments of Pathology,
Internal
Medicine, and
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
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ABSTRACT |
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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 PPAR The transcription factor
PPAR A number of synthetic ligands for PPAR Oxidation of LDL creates unknown PPAR 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 PPAR 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
PPAR 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- 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 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- 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-
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 Synthetic Hexadecyl Azelaoyl Phosphatidylcholine Is a High Affinity
Ligand for PPAR Synthetic azPC Interacts with the Ligand-binding Pocket of
PPAR
We performed the converse experiment where unlabeled rosiglitazone or
azPC was used to displace [3H]azPC bound to immobilized
PPAR azPC Is a Potent Agonist for PPAR-responsive Elements--
azPC
was a ligand for PPAR
We compared the concentration-response relationship of azPC and its
acyl analog with rosiglitazone as PPAR azPC Is a Specific PPAR 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.
We determined whether azPC stimulated CD36 transcription by using CD36
promoter-reporter constructs that either contained its PPRE at position
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.
Extracellular azPC Has Ready Access to Intracellular
PPAR Our findings show that oxidatively modified phosphatidylcholines
from oxidized LDL are high affinity ligands and agonists for PPAR PPAR 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 PPAR azPC binding to PPAR 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 PPAR Oxidation of LDL creates phosphatidylcholines with fragmented
sn-2 residues, some with an 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
PPAR 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
PPAR 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 PPAR 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 PPAR
. [3H]azPC bound
recombinant PPAR
with an affinity
(Kd(app)
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 PPAR
or PPAR
revealed that azPC was a specific
PPAR
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 PPAR
that induce
PPAR-responsive genes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,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). PPAR
, 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 PPAR
function is therefore controlled by lipid ligand binding.
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 PPAR
, including
15-deoxy-
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
PPAR
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 PPAR
and PPAR
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).
agonists (10). This process
also creates PPAR
ligands (12, 13), the bulk of which depend on
liberation by phospholipase A2 to free fatty acid oxidation
products (13). PPAR
alters lipid metabolism, enhances lipid
oxidation, and often dampens inflammatory events and signaling pathways
(14, 15). PPAR
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).
(19, 20), and CD36 is induced by PPAR
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.
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 PPAR
, and because these are found in
oxidized LDL, they may contribute to its biologic effects.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-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).
3 Prime, Inc., Boulder, CO), and purified by CsCl gradients. The
His6-tagged PPAR
was constructed similarly using
the M13 primer in pCR2.1 that contained full-length PPAR
1 and a
primer (5'-CTA ATG ATG ATG
ATG ATG ATG GTA CAA GTC CTT GTA
G-3') containing the His6 tag sequence. PPAR
and PPAR
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.
-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 PPAR
was
determined with a carboxy His6-tagged molecule. HeLa cells
were transfected with pCR3.1-PPAR
-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
PPAR
-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 PPAR
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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-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- -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.
-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.
--
We experimentally determined whether a complex
lipid like azPC could function as a ligand for PPAR
. We synthesized
[3H]hexadecyl azelaoyl phosphatidylcholine
([3H]azPC) and incubated it with full-length recombinant
human PPAR
1. The PPAR
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 PPAR
, and that this binding was
dependent on the concentration of immobilized hPPAR
1. We
next varied the concentration of [3H]azPC to establish
its apparent affinity for PPAR
under these conditions. We found that
the binding of [3H]azPC was
concentration-dependent and that its apparent affinity was
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
PPAR
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 PPAR
1.
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Fig. 3.
azPC is a high affinity ligand for
PPAR . a,
[3H]azPC binds to immobilized full-length PPAR
.
Lysates were prepared from HeLa cells transiently transfected with
plasmid pCR3.1-PPAR
-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
PPAR
as a function of azPC concentration.
hPPAR
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
PPAR
1-His6 was determined as in the above
experiments.
--
Rosiglitazone co-crystallizes with the ligand-binding
domain of PPAR
(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 PPAR
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 PPAR
ligand-binding pocket. a, azPC displacement of
[3H]rosiglitazone from PPAR
.
[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
PPAR
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 PPAR
ligand. Competitive displacement of
[3H]rosiglitazone from Ni+-bound PPAR
was
determined with an excess (6.7 µM) of the stated
compound. c, azPC binds to immobilized PPAR
and is
equally displaced by rosiglitazone or unlabeled azPC. The stated
concentration of azPC or rosiglitazone was incubated with
His6-PPAR
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 PPAR
was analyzed as in b with an excess (6.7 µM)
of the stated lipid. Neg, negative.
. We found (Fig. 4c) that unlabeled rosiglitazone
displaced nearly all of the [3H]azPC from PPAR
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 PPAR
ligand, and we found it to be 10-100-fold
less potent as a competitor (not shown).
, 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-
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 PPAR
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- -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-
-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-
-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.
agonists. CV-1 cells were
transfected with the ACox reporter and SV40-
-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.
Agonist--
We determined the
specificity of azPC as a PPAR
agonist by transfecting CV-1 cells
with PPAR
or PPAR
expression plasmids in addition to the ACox
reporter and an SV40
-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 PPAR
-selective
agonist WY14643 or the 5-fold increase induced by azPC in these control cells. When PPAR
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
PPAR
failed to enhance the existing response to either azPC or
rosiglitazone. The ectopic PPAR
was functional because the
PPAR
-selective agonist WY14643 enhanced expression in cells
overexpressing PPAR
and not in cells transfected with PPAR
.
View larger version (15K):
[in a new window]
Fig. 6.
azPC is a selective PPAR
agonist. CV-1 cells were transfected with acyl-CoA
oxidase-PPRE-luciferase and SV40-
-galactosidase without or with
co-transfection with PPAR
or PPAR
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
-galactosidase was determined as
before. These results are representative of two separate
experiments.
View larger version (31K):
[in a new window]
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 PPAR . 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-
-galactosidase, and some
cells were additionally transfected with expression plasmids encoding
RXR
, PPAR
, or PPAR
. The cells were then treated for 16 h
with 1 µM rosiglitazone or azPC before the ratio of
luciferase to
-galactosidase was determined and normalized to
buffer-treated cells. These results are representative of a separate
experiment.
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, PPAR
, or both RXR and PPAR
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 PPAR
, 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 PPAR
. This augmentation only occurred when the
reporter construct contained the PPAR-responsive element, so activation
of PPAR
, and not PPAR
, by azPC stimulates CD36 expression via
this PPRE.
View larger version (18K):
[in a new window]
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- -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
-galactosidase was determined as before, and this
experiment represents the results of an independent experiment.
--
Our results suggest that azPC is a direct agonist for
PPAR
, 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 PPAR
. 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
PPAR
.
View larger version (31K):
[in a new window]
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
.
We find that there is a distinct selectivity for an sn-1
ether bond in both binding to PPAR
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.
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 PPAR
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.
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 PPAR
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 PPAR
, where the
Kd ranges from 40 (33) to 100 (34) to 325 nM (2).
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 PPAR
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.
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 PPAR
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.
-carboxylate function (40,
41). These oxidation products are found in human plasma (42) and atherosclerotic lesions (43). PPAR
, in contrast to PPAR
, 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
PPAR
function (12). However, PPAR
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 PPAR
. By contrast, we show PPAR
to
directly bind intact, and only intact, oxidized phospholipid.
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.
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.
. 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 PPAR
(20) are all present in atherosclerotic lesions.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Elizabeth Meade for supplying the
acyl-CoA oxidase-luciferase reporter, pCR2.1-PPAR, and
pCR2.1-PPAR
. 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-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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