From the Lipoprotein and Atherosclerosis Group, University of Ottawa Heart Institute, Ottawa K1Y 4W7, Canada
Received for publication, December 19, 2002
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ABSTRACT |
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The The Despite the diverse and biologically important functions of LRP,
relatively little is known about the regulation of LRP gene expression.
The human LRP gene consists of 89 exons spanning 92 kb, encoding an
mRNA of 15 kb (26). Although the coding regions of LRP and the low
density lipoprotein receptor share some homology, there is little
apparent similarity in their promoter regions. A portion of the
5'-flanking region of the LRP gene has been previously described (27,
28). In Chinese hamster ovary cells, the minimal promoter driving
expression of the LRP gene was shown to be in a 1.6-kb GC-rich fragment
that does not contain a classical TATA box. An Sp1 sequence at We have studied the regulation of LRP gene expression during human
preadipocyte differentiation and in response to free fatty acid
availability. LRP mRNA was absent in human preadipocytes, and
the appearance of LRP mRNA during differentiation coincided with
that of the peroxisome proliferator-activated receptor We report here that functional cell surface LRP is increased by PPAR Human Preadipocyte Isolation and Culture--
Subcutaneous
adipose tissue was collected from healthy normolipemic subjects
undergoing reduction mammoplasty procedures. Preadipocytes were
isolated and cultured from adipose tissue through collagenase
digestion, centrifugation, and filtration as previously described
(38-41). The preadipocytes were cultured in differentiation media for
10-14 days. The cells were then insulin-starved in the presence or
absence of varying concentrations of the PPAR Cell Culture--
The human liposarcoma cell line SW872
(American Type Culture Collection, Manassas, VA) was previously
characterized and has been shown to be a good cell model for adipocyte
gene expression (42-44). The cells were cultured in Dulbecco's
modified Eagle's medium/Ham's F-12 medium (3:1) (Invitrogen)
supplemented with 5% fetal bovine serum and 1%
L-glutamine (Invitrogen), 10 mM
NaCO3, and 50 µg/ml gentamycin (NovoPharm, Toronto,
Canada) in the presence of 5% CO2 at 37 °C.
Lipoprotein-deficient fetal calf serum was prepared as described
previously (45) and dialyzed against PBS for 24 h. The effect of
fatty acids and thiazolidenediones on LRP mRNA and protein levels
was determined by incubating cells for 24 h in medium containing
either lipoprotein-deficient fetal calf serum or CS in the presence or
absence of oleic acid (18:1) or arachidonic acid (20:4) (Sigma) or
rosiglitazone (Smith Kline Beecham Pharmaceuticals, King of Prussia,
PA). All of the conditions were studied in triplicate.
125I- Transcription Assay--
To determine the transcriptional effect
of rosiglitazone, 500 nM of this ligand was added to cells
cultured as described above in the presence or absence of 10 µg/ml
RNA Extraction, Northern Blot, and RT-PCR--
Total
cellular RNA was isolated from both differentiated primary human
adipocytes and SW872 cells with Tri-Reagent (Bio/Can, Mississauga,
Canada) according to the manufacturer's instructions. RNA samples from
differentiated primary adipocytes that were to be used in RT-PCR
reactions were treated with amplification grade DNase I to deplete the
samples of any DNA contamination according to the manufacturer's
instructions (Invitrogen). RNA concentration was determined
spectrophotometrically using
A260/280.
Total RNA (5 µg) was separated by agarose gel electrophoresis using
the NorthernMax-Gly kit and transferred to BrightStar-Plus nylon
membrane according to the manufacturer's instructions (Ambion, Austin,
TX). DNA probes were synthesized by RT-PCR; first strand DNA was
synthesized as described below, and PCR was performed using the
following primers: LRPf, 5'-GAGTACCAGGTCCTGTACATCGCTG-3', and LRPr,
5'-CTCGTCAATCATGCCCGAGATGAGC-3';
RT-PCR was performed using a two-step approach. First strand cDNA
was synthesized using 2.5 µg of total RNA, 10 µM random decamer primers (Ambion), and 200 units of Moloney murine leukemia virus reverse transcriptase (Invitrogen) and incubated at 42 °C for
1 h. Consecutive PCR reactions were then performed on the first
strand cDNA using the primers shown below and the SYBR green "taq-start" polymerase and the LightCycler Apparatus according the
manufacturer's instructions (Roche Molecular Biochemicals). The data
from the LightCycler was repeated using relative quantitative RT-PCR as
described below. For SW 872 samples, relative quantitative RT-PCR was
performed using the Quantum RNA 18 S Internal Standards kit from
Ambion. This kit has been previously shown to allow the accurate
determination of relative changes in gene expression between samples
(48). Briefly, first strand cDNA was synthesized using 2.5 µg of
total RNA, 10 µM random decamer primers (Ambion), and 200 units of Moloney murine leukemia virus reverse transcriptase (Invitrogen) and incubated at 42 °C for 1 h. LRP forward primer (5'-GAGTACCAGGTCCTGTACATCGCTG-3') and reverse primer
(5'-CTCGTCAATCATGCCCGAGATGAGC-3') were designed to amplify a region of
the LRP mRNA that is ~400 bp in size, whereas the primers
provided in the 18 S Internal Standards kit produced a band that is
~500 bp. A cycle number of 23 was determined to be within the linear
range of PCR and was used for all subsequent PCR reactions. The 18 S
primer:competimer ratio of 3:7 was experimentally determined so that
the LRP and 18 S PCR products were amplified to give similar yields so
that they could be compared between samples. PCR was performed on 1 µl of the RT reaction using 20 pmol of each LRP primer and 4 µl of
18 S primer/competitor mix with the following PCR conditions; 1 cycle
of 95 °C for 3 min and 23 cycles of 95 °C for 30 s, 66 °C
for 30 s, and 72 °C for 30 s. Cocktails containing all
shared components were used to reduce variation between samples. The PCR products were subjected to electrophoresis through a 1.5% agarose
gel and visualized with ethidium bromide staining. The band intensities
were measured using the ChemiDoc apparatus and Quantity One software
(Bio-Rad). Relative intensity was calculated by dividing the 400-bp
band corresponding to the LRP message by the 488-bp band corresponding
to the 18 S message.
Cell Surface Fluorescent Detection of LRP--
The cells were
cultured on 35-mm cover glass bottom dishes (MatTek, Ashland, MA) as
described above and supplemented with 160 µM arachidonic
acid, 500 nM rosiglitazone, or control. Western Blotting of LRP--
Total cellular protein (5 µg)
from SW872 cells incubated in the presence or absence of various
PPAR Preparation of Nuclear Protein Extracts--
The nuclear
proteins were extracted from SW872 cells as previously described (51)
or from primary human adipocytes as described (52). Protein
concentration was determined using BCA protein reagent (BioLynx,
Brockville, Canada) according to the manufacturer's instructions.
Electrophoretic Mobility Shift Assays
(EMSA)--
Double-stranded oligonucleotides (oligomers) corresponding
to the PPRE of LRP
(5'-CCCCGCTCCTTGAACTCTGACATCGAGACACCTA-3') were radioactively end-labeled with [
Supershift assays were performed using the PPAR LRP Reporter Gene Constructs--
Complementary primers with
flanking NheI and XhoI restriction sites
(5'-CTAGCCTCCTTGAACTCTGACATGCAGACC-3')
were annealed and subcloned into the luciferase reporter vector,
pGL3-Promoter (Promega). This new vector contains a single copy of the
putative PPRE upstream of an ideal promoter and is designated
pGL3-PPRE.
We also prepared 1.9 kb of the 5'-flanking region of LRP by PCR
amplification from the LRP-BAC construct prepared by Dr. Jan Boren
(53), which contains the entire 92-kb gene of human LRP, using the
primers 5'-GCAACGAGCTCCGTAAAAGGGGGAAG-3' and
5'-GCAGCAGATCTTTCCCCGGACTGAAG-3'. This fragment was subcloned into the
SacI and BglII sites of the luciferase reporter
vector, pGL3-Basic (Promega), and designated pGL3-LRP. Mutagenesis of
the PPRE was performed by PCR using PFUTurbo (Stratagene, La Jolla, CA)
according to their Quikchange site-directed mutagenesis protocol. The
complementary primers
(5'-CCCGCTCCTTGAACTCAACGATGCAGACACC-3') were designed to
mutate a single half-site of the PPRE so that PPAR Transient Transfection Assays--
Confluent SW872 cells were
trypsinized and seeded at a density of 1.25 × 105
cells/well in 12-well plates 48 h prior to transfection. The cells
were ~70-80% confluent at the time of transfection. Fresh medium
containing CS was added 12 h preceding transfections. The cells
were co-transfected with 4 µg of the firefly luciferase reporter
vector (either pGL3-basic, pGL3-LRP, PGL3-PPRE, or
pGL3-LRPmutant PPRE) and 0.25 µg of the
Renilla luciferase-bearing reporter vector, pRL-CMV
(Promega) using the calcium phosphate-DNA precipitate method (54). The
cells were shocked with 15% glycerol for 2 min, 4 h after the
transfection, and washed three times with PBS before the addition of
medium. The cells were treated 12 h later with Me2SO
alone (vehicle control) or varying concentrations of rosiglitazone.
After 24 h (36 h total post-transfection) the cells were scraped
in 250 µl of reporter lysis buffer (Promega) and kept on ice until assayed.
Luciferase activities derived from both firefly (LRP constructs) and
Renilla (pRL-CMV) proteins were measured using the dual luciferase reporter assay system (Promega) and recorded using a
Monolight 2010c luminometer (Analytical Luminescence Laboratory, Ann
Arbor, MI). Renilla luciferase activity was then used to
standardize for transfection efficiency.
Statistical Analysis--
The results are expressed as the
means ± S.E. Where indicated, the statistical significance of the
differences between groups was determined using Student's t
test or analysis of variance.
Functional Cell Surface LRP Is Increased upon Exposure to PPAR
The increase in functional cell surface LRP was confirmed in SW872
cells by cell surface labeling experiments using fluorescently labeled
Endogenous LRP mRNA Levels Are Increased upon Exposure to
PPAR
The effect of rosiglitazone on LRP mRNA levels was also determined
in differentiated primary human adipocytes, as measured by Northern
blot and real time PCR, and these results are summarized in Fig.
5. According the Northern blot analysis,
LRP mRNA is increased by rosiglitazone treatment by ~1.6-fold.
Using real time RT-PCR, there was a dose-dependent increase
in LRP mRNA levels (ranging from 1.4- to 1.8-fold) after 24 h
of ligand treatment. The levels of LRP mRNA were also verified
using relative quantitative RT-PCR, and the fold increases in LRP
expression were similar to those shown in Fig. 5.
PPAR PPAR
The shift caused by the nuclear extracts was further analyzed by gel
supershift analysis to confirm that PPAR LRP PPRE Luciferase Constructs Are Responsive to Rosiglitazone in
Dual Luciferase Assay--
Although the basal transcriptional activity
of the pGL3-PPRE construct was ~2-fold higher than the pGL3-LRP
construct because of the context of the SV40 ideal promoter (data not
shown), the promoter context of the endogenous LRP promoter, which is
present in pGL3-LRP, is the more physiologically relevant of the two
constructs. This portion of the promoter drove the basal activity of
the reporter gene as determined by the dual luciferase reporter assay
system (Fig. 7A). The values
were normalized to the cells treated with Me2SO only
(vehicle control). The empty pGL3-basic vector had no basal activity
above the background measurements of the instrument (data not shown).
For pGL3-LRP (Fig. 7A), as well as pGL3-PPRE (data not
shown), there was a dose-dependent response of the
luciferase activity upon treatment with rosiglitazone that corresponded
to the increases in LRP mRNA shown in Fig. 4. The ratio of firefly and Renilla luciferase activities are shown in the figures;
it is important to note that the luciferase activity increases with rosiglitazone treatment and that the Renilla does not
decrease.
Mutagenesis of the PPRE Results in the Loss of Enhancer
Activity--
A single half-site of the PPRE, to which PPAR Despite the importance of LRP in lipoprotein, serum protease, and
As anticipated, there was an inverse correlation between the amount of
ligand required and the affinity of the ligand for PPAR Previous studies have examined the effect of sterols on LRP
transcription and reported that LRP, unlike the low density lipoprotein receptor and other members of this receptor family, was not
down-regulated by sterols (27). Further study identified a sequence
corresponding to a sterol response element in the 5'-untranslated
region of the LRP transcript (28), which appears inactive because LRP does not show any response to sterols. This is in agreement with our
data demonstrating that LRP mRNA levels are similar when cells are
cultured in CS as compared with lipoprotein-deficient fetal calf serum.
PPAR LRP is expressed in various tissues (23), and its function in each cell
type differs widely. Thus, the regulation of LRP gene expression and
function by insulin-sensitizing agents of the glitazone class or by
fibrates could have considerable clinical importance. Three PPAR
subtypes ( We have demonstrated that adipocyte LRP expression and function is
up-regulated by rosiglitazone, a widely used insulin-sensitizing agent.
Rosiglitazone has been shown to enhance plasma triglyceride clearance
by an unknown mechanism (75), which we hypothesize may involve
adipocyte LRP. Fibrates substantially decrease plasma triglyceride
levels, and this effect has been primarily attributed to an increase in
lipoprotein lipase activity and decreased expression of apoCIII (33,
34, 75). We propose that a PPAR2-macroglobulin
receptor/low density lipoprotein receptor-related protein (LRP) is a
large multifunctional receptor that interacts with a variety of
molecules. It is implicated in biologically important processes such as
lipoprotein metabolism, neurological function, tissue remodeling,
protease complex clearance, and cell signal transduction. However, the
regulation of LRP gene expression remains largely unknown. In this
study, we have analyzed 2 kb of the 5'-flanking region of the LRP gene
and identified a predicted peroxisome proliferator response element
(PPRE) from
1185 to
1173. Peroxisome proliferator-activated
receptor
(PPAR
) ligands such as fatty acids and rosiglitazone
increased functional cell surface LRP by 1.5-2.0-fold in primary human
adipocytes and in the SW872 human liposarcoma cell line as assessed by
activated
2-macroglobulin binding and degradation. These
agents were found to increase LRP transcription. Gel shift analysis of
the putative PPRE demonstrated direct binding of PPAR
/retinoid X
receptor
heterodimers to the PPRE in the LRP gene. Furthermore,
these heterodimers could no longer interact with a mutated PPRE probe. The isolated promoter was functional in SW872 cells, and its activity was increased by 1.5-fold with the addition of rosiglitazone. Furthermore, the isolated response element was similarly responsive to
rosiglitazone when placed upstream of an ideal promoter. Mutagenesis of
the predicted PPRE abolished the ability of this construct to respond
to rosiglitazone. These data demonstrate that fatty acids and
rosiglitazone directly stimulate transcription of the LRP gene through
activation of PPAR
and increase functional LRP expression.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2-macroglobulin receptor/low density
lipoprotein receptor-related protein
(LRP)1 is a 600-kDa
multifunctional endocytic receptor that belongs to the low density
lipoprotein receptor gene family (1). LRP binds and internalizes a
broad range of biologically diverse ligands. These include proteases of
the fibrinolytic pathway (2) and serpin-enzyme complexes (3) as well as
proteins important in lipoprotein metabolism such as lipoprotein
lipase, hepatic lipase, lipoprotein(a), and apoE-rich lipoproteins
(4-9). Targeted deletion of LRP in the mouse results in early
embryonic death, demonstrating a critical function for LRP in prenatal
development (10). LRP has also been shown to have a dual role in
-amyloid metabolism by enhancing
-amyloid precursor protein
conversion to
-amyloid (11) and mediating the clearance of
-amyloid (12, 13). These data support a potentially complex role for
LRP in the pathogenesis of Alzheimer's disease (14). In addition, LRP
mediates signal transduction by interacting with cytosolic adaptor and
scaffold proteins including DAB-1, JIP-2, and PSD-95 (15). A 39-kDa
receptor-associated protein (RAP) is an endoplasmic reticulum-resident
protein that functions intracellularly as a molecular chaperone for LRP
and regulates its ligand binding activity (16-18). RAP is required for
the proper folding and export of the LRP from the endoplasmic reticulum
by preventing the premature binding of co-expressed ligands, such as
apoE (19-21). RAP binds LRP directly via adjacent complement-type
repeats, both containing a conserved acidic residue (22), and thus
stearically interferes with binding of other LRP ligands including
2M* and remnant lipoproteins. LRP is expressed in a variety of cells
with high expression in hepatocytes, macrophages, neuronal cells,
fibroblasts, and adipocytes (23). In human adipocytes, LRP is involved
in chylomicron remnant cholesterol clearance (24) and mediates the
selective uptake of high density lipoprotein-derived cholesteryl ester
(25).
80 and
two clusters of Sp1 sequences between
520 and
752 were
characterized and shown to be critical for expression of the gene. The
promoter region also contains a consensus NRF-1 element located at
152 that may mediate the effects of cAMP and IFN
(29, 30). There
is a consensus sterol response element located at +233 in the
5'-untranslated region; however, studies have shown that LRP gene
expression is not regulated by cholesterol (27).
(PPAR
).2 PPAR
is a
transcription factor belonging to the nuclear hormone receptor
superfamily. The retinoid X receptor
(RXR
) is the obligate
partner of PPAR
(31), and together they form a heterodimer that
regulates gene transcription following binding to a peroxisome proliferator response element (PPRE) and activation by specific ligands. The PPRE consists of a hexameric nucleotide repeat of the
recognition motif (TGACCT) spaced by one nucleotide (DR-1) (32, 33).
PPAR
is activated by a number of ligands including long chain fatty
acids (34), prostaglandin J2 derivatives (35), and thiazolidenediones
(36, 37). The effects of PPAR
ligands on gene expression are direct
results of increased transcription of the target gene containing a
PPRE. In our own analysis of the LRP promoter, we have identified a
novel sequence
(TGAACTcTGACAT) in the
5'-flanking sequence at positions
1185 to
1173 with high homology
to the PPRE.
ligands via the activation of PPAR
transcriptional complexes that
bind the newly identified PPRE in the LRP promoter. This is the first
report demonstrating regulation of LRP gene expression via a discrete
promoter element.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ligand rosiglitazone
for 24 h prior to assays. The control cells were treated with
vehicle only (Me2SO).
2M Degradation and Binding Assays--
SW872
cells were cultured for 24 h with either 160 µM
arachidonic acid or 500 nM rosiglitazone in media
containing CS prior to measuring their ability to degrade
125I-labeled
2M* as previously described (46).
Differentiated primary human adipocytes cultured in differentiating
medium were starved of insulin in the presence or absence of increasing
concentrations of rosiglitazone prior to measuring their ability to
degrade or bind 125I-
2M* (46). Control cells were
treated with vehicle only (Me2SO). As a further control,
the cells were treated in the presence or absence of RAP because this
molecule will impair LRP function.
-amanitin (Sigma), a potent inhibitor of RNA polymerase II (47).
-actin-f,
5'-GCCCCTCCATCGTCCACCGC-3', and
-actin-r,
5'-GGGCACGAAGGCTCATCATT-3'. The PCR products were gel-purified using
the QiaexII kit (Qiagen), and the purified DNA was subsequently labeled
using the Rediprime II random prime labeling kit according the
manufacturer's instructions (Amersham Biosciences). The probes were
cleaned up with NICK columns (Amersham Biosciences), and the specific
activity was determined by use of a scintillation counter.
Hybridizations and washes were performed according the NorthernMax-Gly
kit instructions (Ambion).
2M was activated
by incubating purified
2M with 400 mM methylamine for
16 h at room temperature.
2M* was fluorescently labeled using Cy3 monofunctional reactive dye (Amersham Biosciences) to a dye:protein ratio of 1.3 according to the manufacturer's instructions. The cells were placed on ice for 1 h in 3:1 Dulbecco's modified
Eagle's medium/Ham's F-12 medium supplement with 2 mg/ml BSA buffered with 10 mM HEPES. Labeled
2M* was diluted in the same
medium to a concentration of 1 µg/ml and was added to the cells at
0 °C for 45 min. The cells were washed with ice-cold PBS three times prior to being fixed with 4% paraformaldehyde for 10 min at 0 °C.
The cells were rinsed with PBS and kept in 2 ml of PBS at room
temperature for fluorescence microscopy. Binding was competed with
30-fold excess of unlabelled
2M*. The cells were viewed with an
Olympus IX50 fluorescent microscope, and the images were taken using a
coded CCD camera (MicroMax) and WinView software from Princeton
Instruments (Princeton, NJ).
ligands was subjected to SDS-polyacrylamide gel electrophoresis
and transferred to nitrocellulose (49). The LRP was detected (49) using
a polyclonal rabbit antisera (from Dr. G. Bu) followed by
chemiluminescent detection (Pierce) of a secondary antibody conjugated
to horseradish peroxidase. The blot was developed, and the bands were
quantified using the ChemiDoc apparatus and Quantity One software
(Bio-Rad). Triplicate cell samples were processed and are summarized in
the graph. Molecular biology techniques were essentially as described
by Sambrook et al. (50).
-32P]dATP (Amersham
Biosciences) using T4 polynucleotide kinase (Invitrogen) and purified
from unincorporated nucleotides by gel filtration through G-50 spin
columns (Amersham Biosciences). The same procedure was used for
oligomers corresponding to the PPRE of the human fatty acyl CoA oxidase
gene (hACOX) (5'-TCCGAACGTGACCTTTGTCCTGGTCCCCTTT-3') and
oligomers corresponding to the mutated form of the LRP PPRE (the
mutated half-site is underlined)
(5'-CCCCGCTCCTTGAACTCAACGATCGAGACACC TA-3'). The specific activities of the oligomers were ~250 cpm/fmol. These were diluted to 60 fmol/µl for use in the assay. PPAR
(from Dr. Bruce Spiegelman) was subcloned into the MluI and
NotI sites of pSPORT1 (Invitrogen) using PCR-based methods.
RXR
was provided by Dr. Michael Saunders in pSG5. Both constructs
are driven by the T7 RNA polymerase promoter for use in the TNT
T7-coupled reticulocyte lysate system (Promega) for in vitro
transcription/translation. All of the EMSA reactions were carried out
on ice in 20 µl of binding buffer (12.5 mM HEPES-KOH, pH
7.6, 6 mM MgCl2, 5.5 mM EDTA, and
50 mM KCl) supplemented with 5 mM
dithiothreitol, 0.25 µg of low fat milk, 0.05 µg of poly(dI-dC),
and 10% glycerol. For EMSA reactions with TNT-purified proteins, 2 µl of the TNT reaction was added to the reaction mix along with 1 µl (60 fmol) of labeled oligomers. For EMSA reactions with nuclear
protein extracts, 6 µl of nuclear extracts were added to the reaction mix with 1 µl (60 fmol) of labeled oligomers. These reactions were
left on ice for 20 min. Following the 20-min incubation, 2 µl of 20%
Ficoll was added to the samples. DNA-protein complexes were then
resolved by electrophoresis through 6% polyacrylamide gels in 0.25×
Tris borate running buffer (20 mM Tris borate, pH 7.2, 0.5 mM EDTA).
NuShift kit
following the manufacturer's protocol (Active Motif). The nonspecific antibody used was mouse monoclonal anti-
-actin (Santa-Cruz).
would no longer
bind the response element (mutated nucleotides are underlined). This
construct was designated pGL3- LRPmutant PPRE. Both
pGL3-LRP and pGL3-LRPmutant PPRE were sequenced to confirm
that the promoter sequence was correct (compared with
GenBankTM accession number Y18524) and to verify the mutagenesis.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Ligands--
The effects of PPAR
ligands on differentiated primary
human adipocytes were examined by an 125I-
2M* binding
assay (Fig. 1A). The
Bmax of cells incubated with 1 µM
rosiglitazone (27.0) is ~1.5 times greater than that of the vehicle-treated cells (17.3), indicating that there is an increase in
the levels of functional cell surface LRP. The difference in binding
was found to be highly significant with a two-tailed p value
of less than 0.0001. When the cells were treated with RAP (30 µg/ml),
the amount of binding was reduced to background levels, demonstrating
that this process is LRP-specific. There was no statistically
significant difference in the Kd between the treated
and control cells as illustrated in the Scatchard plot (Fig.
1B), indicating that the binding affinities have not changed. 125I-
2M* degradation assays were also performed
in the presence or absence of PPAR
ligands for the differentiated
primary human adipocytes and SW872 cells. In primary adipocytes, there
was a direct relationship between the amount of rosiglitazone added and
the amount of
2M* degradation over 8 h (Fig.
2A) with very significant
increases ranging from 1.2- to 1.7-fold over control (p < 0.009). The RAP is an antagonist of all identified LRP ligands including
2M*; therefore we used purified RAP to block LRP function in our assays. When cells were treated with RAP, the amount of 125I-
2M* degraded was diminished to background levels,
demonstrating that this process is LRP-specific. Degradation assays
were also performed in the SW872 cells treated with rosiglitazone (Fig. 2B) or arachidonic acid (Fig. 2C). There was a
1.5-fold increase in the amount of 125I-
2M* degraded
over 8 h in the treated cells versus the control cells
for both of the PPAR
ligands.
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Fig. 1.
Increased cell surface binding of
125I- 2M* by cells treated with a
PPAR
ligand. A,
125I-
2M* binding curve for differentiated primary human
adipocytes. Differentiated primary human adipocytes were preincubated
for 24 h in differentiating medium without insulin in either 1 µM rosiglitazone or vehicle alone (Me2SO).
The cells were then washed twice with HBSS, 25 mM
HEPES, pH 7.45, at 37 °C for 20 min each and placed on ice for 20 min to allow the cells to equilibrate, and then ice-cold HBSS, 25 mM HEPES, pH 7.45, 10 mg/ml BSA containing various
concentrations of 125I-
2M (1, 0.5, 0.25, 0.125, 0.0625, and 0.03125 mg/ml) was added to the cells. The cells were also
incubated in the presence or absence of RAP (30 µg/ml), a LRP ligand
that acts as an antagonist to the binding of all other LRP ligands. The
cells were further incubated on ice for 2 h and then washed six
times with ice-cold HBSS, 25 mM HEPES, pH 7.45. the cells
were then solubilized in 0.2 M NaOH and then cell
associated radioactivity counted. B, Scatchard analysis of
125I-
2M* binding curves. Scatchard analysis was
performed on the data plotted in A to obtain information
regarding the binding kinetics of 125I-
2M on
differentiated primary human adipocytes in the presence of vehicle
alone or 1 µM rosiglitazone. This analysis is presented
as a Scatchard plot with bound/free plotted against the bound
125I-
2M*. The Kd is a measure of the
binding affinity between the ligand and the receptor and is determined
from the slope. The Bmax is a measure of the
number of receptors on the cell surface and is represented by the
x intercept. The data points represent means from
triplicate experiments, and the error bars represent the
standard error of the mean. *, the two-tailed p value is
<0.0001.
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Fig. 2.
Increased cellular degradation of
125I- 2M* by cells treated with
PPAR
ligands. A,
differentiated primary human adipocytes were treated with various
concentrations of rosiglitazone in the absence of insulin for 24 h
prior to the experiment. The cells were then incubated at 37 °C with
HBSS, 25 mM HEPES, pH 7.45, 10 mg/ml BSA containing 1 mg/ml
125I-
2M* with or without RAP (30 µg/ml). At the
beginning of the incubation period and at each time point, the medium
was precipitated with ice-cold tricarboxylic acid, and the
tricarboxylic acid-soluble material was used as a measure of the
degraded protein. The data points represent the means from
triplicate experiments, and the error bars represent the
standard error of the mean. *, p = 0.009; **,
p = 0.007; ***, p = 0.002. B
and C, SW872 cells (treated with vehicle, 500 nM
rosiglitazone, and 160 µM arachidonic acid) were washed
at 37 °C to remove any residual fetal calf serum. The cells were
incubated at 37 °C for the indicated times in HBSS, 25 mM HEPES, pH 7.45, 10 mg/ml BSA containing 1 mg/ml
125I-
2M*. At the beginning of the incubation period and
at each time point, the medium was precipitated with ice-cold
tricarboxylic acid, and the tricarboxylic acid-soluble material was
used as a measure of the degraded protein. Trichloroacetic acid-soluble
material from untreated cells (vehicle) was compared with the
trichloroacetic acid-soluble material from cells treated with 500 nM rosiglitazone (B) or 160 µM
arachidonic acid (C). The data points represent
means from triplicate experiments, and the error bars
represent the standard error of the mean. *, p = 0.006;
**, p = 0.03. DMSO, dimethyl
sulfoxide.
2M* in the presence of 160 µM arachidonic acid or 500 nM rosiglitazone (Fig.
3A). In addition, total
cellular LRP was increased in those cells as determined by Western blot
analysis (Fig. 3B). The increases seen in LRP using these
methods were ~1.5-2-fold, supporting the binding and degradation
data above.
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Fig. 3.
LRP protein levels are increased in SW872
cells upon exposure to PPAR ligands.
A, cells were incubated with 1 µg/ml fluorescently labeled
activated
2M (
2M*) (Cy3 monofunctional reactive dye) on ice for
45 min in 3:1 Dulbecco's modified Eagle's medium/Ham's F-12 medium
supplement with 2 mg/ml BSA buffered with 10 mM HEPES after
an equilibration period of 1 h. The cells were washed with
ice-cold PBS three times prior to being fixed with 4% paraformaldehyde
for 10 min at 0 °C. The cells were rinsed with PBS and kept in 2 ml
of PBS at room temperature for fluorescence microscopy. The photographs
are representative of cells treated with the ligands indicated. A
30-fold excess of unlabeled
2M* was used to compete for binding with
the fluorescently labeled
2M*. The photographs are normalized so
that an increase in intensity on the cell surface and the cell
circumference between photographs represents an increase in the
fluorescence (i.e. total binding). B, Western
blot of cells treated with Me2SO (vehicle control), 160 µM arachidonic acid, or 500 nM rosiglitazone
for 24 h. 5 µg of total protein was loaded in each lane. The
blot was developed using chemiluminescent techniques, and the bands
were visualized using the ChemiDoc apparatus. The band at 515 kDa
corresponds to the
-subunit of LRP. C, quantification of
Western blot. The 515-kDa band corresponding to LRP was quantified
using Quantity One software. All of the samples were normalized to the
vehicle-treated cells (control). The Results are shown as the means of
triplicate experiments, and the error bars represent the
standard error of the mean. *, the two-tailed p value is
0.002; **, the two-tailed p value is 0.003.
Ligands--
The effect of PPAR
ligands on levels of LRP
mRNA was determined in the adipocytic cell line, SW872, by relative
quantitative RT-PCR. The relative intensity was determined by the ratio
of the LRP band intensity compared with the 18 S band intensity, and
these values were normalized to the control samples to give values of
fold increase. The fold increases of LRP mRNA in cells upon
treatment with oleic acid, arachidonic acid, and rosiglitazone are
summarized in Fig. 5 below (A, B, and
C, respectively). PPAR
ligands, rosiglitazone (500 nM), arachidonic acid (160 µM), and oleic
acid (0.8 mM) increased LRP mRNA levels by 1.5-, 1.6-, and 1.3-fold, respectively. The maximum effect of the ligands was observed after 24 h of treatment. Increases in LRP mRNA levels were not observed for concentrations of rosiglitazone above 1 µM (data not shown). When cells were cultured in medium
containing lipoprotein-deficient fetal calf serum instead of CS, the
increases in LRP mRNA levels were similar to those shown in Fig.
4 (B and C).
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Fig. 4.
Endogenous LRP mRNA levels in SW872 cells
are modulated by PPAR ligands at the
transcriptional level. The effect of PPAR
ligands on levels of
LRP mRNA was determined by incubating SW872 cells for 24 h in
Dulbecco's modified Eagle's medium/Ham's F-12 medium (3:1)
supplemented with complete serum in the presence or absence of the
PPAR
ligands. A, oleic acid. B, arachidonic
acid. C, rosiglitazone. Also a potent inhibitor of RNA
polymerase II,
-amanitin, was added to SW872 cells in the presence
or absence of 500 nM rosiglitazone to inhibit transcription
(C). Reverse transcription was performed on total RNA
isolated from these cells, and multiplex PCR was performed using LRP
and 18 S gene-specific primers (relative quantitative RT-PCR). PCR
products were visualized by EtBr staining on the ChemiDoc, and the band
intensities were determined using Quantity One software. The intensity
of the LRP product was divided by the intensity of the 18 S product to
obtain a value termed relative intensity. These results were normalized
to the control and shown as fold increases. The bars in each
graph represent the means of triplicate experiments, and the
error bars represent the standard error of the mean.
One-tailed p values from paired t test are shown
above the bars to indicate significant differences.
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Fig. 5.
Endogenous LRP mRNA levels in
differentiated primary human adipocytes are modulated by
rosiglitazone. A, Northern blot analysis of total RNA
from differentiated primary human adipocytes. Differentiated primary
human adipocytes were cultured for 24 h in differentiating medium
without insulin in the presence or absence of 1 µM
rosiglitazone Total RNA isolated and separated by agarose gel
electrophoresis and transferred to BrightStar-Plus nylon membrane. The
membrane was probed with either an LRP or -actin random labeled
probe. A representative blot is shown here. B,
quantification of Northern blots. Northern blots were scanned using the
ChemiDoc, and densitometry was performed using Quantity One software.
The data shown are the averages of three independent Northern blots
from the RNA of three individual tissue samples (each done in
triplicate), and the error bars represent the standard error
of the mean. C, quantification of mRNA changes using
real time RT-PCR. Differentiated primary human adipocytes were cultured
for 24 h in differentiating medium without insulin in the presence
or absence of various concentrations of rosiglitazone. Reverse
transcription was performed on total RNA isolated from these cells, and
independent real time PCR reactions were performed using LRP and 18 S
gene-specific primers on the LightCycler. These results were normalized
to the control and shown as fold increases. The bars in each
graph represent the means of triplicate experiments, and the
error bars represent the standard error of the mean. The
same RT reaction was subjected to relative quantitative RT-PCR, and the
results were similar to those shown here. Two-tailed p
values from Student's t test are shown above the
bars to indicate significant differences. A p
value of 0.02 for all of the values is obtained using analysis of
variance.
Ligands Act at the Transcriptional Level to Increase LRP
mRNA--
The increase in LRP mRNA could be due to an mRNA
stabilization effect or to increased transcription of the mRNA. To
distinguish between these possibilities, a potent inhibitor of RNA
polymerase II activity,
-amanitin, was used to inhibit new
transcriptional activity. If the level of mRNA were increased by a
stabilization effect of rosiglitazone or other PPAR
ligands, then
the increase in mRNA levels would still be observed when both
-amanitin and rosiglitazone were added to cells concomitantly. We
did not observe an increase in LRP mRNA in cells cultured with both
rosiglitazone and
-amanitin (Fig. 4C). Although there was
a small decrease in LRP mRNA levels with the addition of
-amanitin, this decrease was identical in both the vehicle-treated
and rosiglitazone-treated cells, suggesting this is due to normal
turnover of the mRNA. These results support a role for these
ligands as transcriptional up-regulators of LRP gene expression.
-RXR
Heterodimers Selectively Bind the PPRE Identified in
the LRP Promoter Region--
We identified a PPRE located at
1185 to
1173 of the LRP promoter through sequence scanning of the 5'-flanking
region. The sequence homology to the consensus PPRE is 83%; there is a
single mismatch per half-site of the DR-1 (Fig.
6A). The TNT in
vitro transcription/translation of pSPORT1-PPAR
and pSG5-RXR
was first shown to express PPAR
and RXR
; when
[35S]methionine was added to the reaction mix, proteins
of the correct molecular mass were synthesized as verified by SDS-PAGE
and autoradiography. For the EMSA, these constructs were subjected to
the TNT reaction (without [35S]methionine) and incubated
with 32P-end-labeled oligomers corresponding to the PPRE of
LRP. A clear shift was evident in the presence of these transcription
factors (Fig. 6B, lane 2) compared with when the
TNT reaction was carried out with the empty pSPORT1 vector
(unprogrammed lysate) (lane 1). The interaction between the
protein complex and the DNA could be competed by the addition of
increasing amounts of excess of unlabeled hACOX oligomer (lanes
3-6). Identical experiments using the hACOX probe yielded very
similar results (Fig. 6B, lanes 7-12). The
doublet bands that appear have been observed in other studies where
rabbit reticulocyte lysate was used to produce PPAR
and RXR
(55-57). These doublets cannot be homodimers because PPAR
and
RXR
TNT reactions, added individually to the EMSA reaction, did not
give shifts (data not shown). Incubation of the probes with nuclear
protein extracts from primary adipocytes gave a shift of the same size
as the TNT proteins (Fig. 6C, lane 2). This shift could also be inhibited by increasing amounts of excess unlabeled hACOX
oligomer (lanes 2-5). Nuclear extracts from the SW872 cell line yielded similar results. These results are comparable with those
obtained using oligomers corresponding to the hACOX PPRE (lanes
6-10). A probe containing a mutated PPRE half-site,
LRPmut, could no longer interact and bind TNT proteins
(Fig. 6B, lane 14) or nuclear extracts (Fig.
6C, lane 11).
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Fig. 6.
PPAR -RXR
heterodimers selectively bind PPRE in the LRP promoter. EMSA
were performed on oligomers corresponding the PPREs of LRP, hACOX, and
LRPmut. A, the PPREs of the human LRP and ACOX
genes are shown compared with the consensus PPRE along with the
sequence of the mutant LRP PPRE. The mismatches for each PPRE are
underlined. B, oligomers were incubated on ice
for 20 min with various components. Unprogrammed reticulocyte lysate
(TNT reaction containing empty pSPORT1 vector) was used as a control
(lanes 1, 7, and 13). TNT in
vitro transcribed/translated PPAR
and RXR
were incubated
with radiolabeled PPRE oligomers (lanes 2, 8, and
14). To control for specificity of binding competition
experiments were performed by adding increasing amounts of unlabeled
oligomer to the binding reactions. LRP binding reactions were competed
using cold hACOX oligomers at 5×, 10×, 25×, and 50× excess
(lanes 3-6), and the hACOX binding reactions were competed
using cold LRP oligomers at 5×, 10×, 25×, and 50× excess
(lanes 9-12). C, nuclear extracts from primary
human adipocytes were incubated with radiolabeled oligomer (lanes
1, 6, and 11) on ice for 20 min. Specificity
of binding was again tested using unlabeled oligomers in competition
experiments as described above (lanes 2-5 and
7-10). D, gel supershift analysis was used to
determine whether PPAR
was present in shift seen with nuclear
extracts. Free probe (first lane) was used as a control
reaction. The nuclear extracts (18 µg) were incubated with labeled
oligomer and run in the second lane. NuShift anti-PPAR
antibody (4 µl) was incubated with 18 µg of nuclear extracts from
SW872 cells prior to being added to the EMSA reaction mix containing
labeled oligomers (third lane). A nonspecific antibody
(mouse monoclonal anti-
-actin) was used as a negative control for
the supershift (fourth lane). The arrows indicate
the shift and the supershift.
was a component of this
complex (Fig. 6D). Free probe was run in lane 1 as a control reaction, whereas a reaction containing nuclear protein extracts from
primary human adipocytes is shown in the second lane. The nuclear protein extracts were preincubated with anti-PPAR
antibody prior to being added to the reaction containing labeled oligomers, and
this was run in the third lane. A band of the same size as that in the second lane was present as well as a larger band
that was absent in all other lanes (supershift). The same results were obtained for the oligomer corresponding to the hACOX PPRE. A supershift was not observed when a nonspecific antibody was used in place of the
anti-PPAR
antibody. Similar results were also obtained using nuclear
extracts from SW872 cells supporting the involvement of PPAR
in the
protein-DNA complex.
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Fig. 7.
LRP promoter confers regulation of luciferase
activity by rosiglitazone. A, the promoter region of
human LRP (0 to 1200) was cloned into pGL3-Basic. This construct was
then transiently co-transfected into SW872 cells along with the
Renilla luciferase reporter vector pRL-CMV using the
calcium-phosphate precipitation method. Luciferase activities for each
of firefly and Renilla luciferase were determined using the
dual luciferase assay system. The intensity of firefly luciferase is
shown as a function of Renilla luciferase (relative
activity). The relative intensity is normalized to the control (vehicle
treatment) values. The means of triplicate experiments are graphed, and
the error bars represent the standard error of the mean. The
one-tailed p values from paired t tests are shown
for significant differences. B, pGL3-LRP was subjected to
site-directed mutagenesis, in which the half-site of the DR-1 to which
PPAR binds, was mutated so that 5 of 6 nucleotides no longer matched
the consensus. PPREs of pGL3-LRP and pGL3-LRPmutant PPRE
are shown with the consensus sequence for comparison. Point mutations
are underlined, and the mismatches present in endogenous
PPRE are in italics. C,
pGL3- LRPmutant PPRE, was transiently co-transfected
with pRL-CMV into SW872 cells, and their respective luciferase
activities were determined using the dual luciferase assay system. The
relative intensity was calculated, and the means of triplicate
experiments are shown. The values of relative intensity are normalized
to the control (vehicle-treated) cells. The error bars
represent the standard error of the mean. Rosiglitazone has a
significant effect on pGL3-LRP; however, it is not significant for
pGL3-LRPmutant PPRE. The one-tailed p values
from t tests were 0.02 and 0.2, respectively.
binds,
was mutated using the Quikchange mutagenesis protocol (Fig.
7B) and designated pGL3-LRPmutant PPRE. This
construct was sequenced, and the point mutations within the PPRE were
verified. The basal activity of this reporter construct (vehicle
control) was similar to that of the pGL3-LRP construct (Fig.
7C). In the presence of 500 nM rosiglitazone
there was approximately a 1.5-fold increase in promoter activity of the
pGL3-LRP construct. This increase, however, was lost in the
pGL3-LRPmutant PPRE construct, indicating a role for this
PPRE as a transcriptional enhancer.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-amyloid metabolism, this is the first study to demonstrate that LRP
expression is regulated at both mRNA and protein levels via a
discrete promoter element. We have demonstrated that functional LRP
expression is regulated by PPAR
ligands by a mechanism that involves
the ligand-induced up-regulation of transcription via the activation of
PPAR
-RXR
heterodimers that bind a newly identified PPRE in the
promoter of the LRP gene. Levels of functional cell surface LRP were
measured by binding and degradation of a well characterized LRP ligand,
2M* (58, 59). Furthermore, we have demonstrated that LRP mRNA
levels are modulated at the transcriptional level by ligands that
activate PPAR
and that this response is dose-dependent.
The increase in LRP mRNA levels with rosiglitazone was shown to
result from direct binding of PPAR
-RXR
heterodimers to the PPRE
identified in the promoter.
(36, 60).
Rosiglitazone, the most potent ligand used, was found to have a maximal
effect at ~500 nM in SW872 cells, although there was
significant up-regulation of LRP at 50 nM, a concentration
much closer to the reported Kd of 40 nM
(36). Concentrations of 750 nM or higher did not alter LRP mRNA abundance (data not shown) or promoter activity of pGL3-LRP in
SW872 cells. In differentiated human adipocytes, LRP mRNA levels were up-regulated by rosiglitazone in a dose-dependent
manner at concentrations up to 1 µM, and the decreased
efficacy was observed only at a concentration of 2 µM. It
has been suggested that rosiglitazone might act as a partial antagonist
at these high concentrations (61). In addition, activation of PPAR
at the AF2 domain enhances its degradation (62), which would explain
the reduced efficacy of rosiglitazone at higher concentrations (Figs. 5
and 7A). In both cell types, LRP expression and function
were increased by 1.5-2-fold, similar to that reported for other genes
containing a PPRE in the promoter region (57, 63, 64). For example, the
PPAR
agonist fenofibrate increases apoA-I expression by
1.5-2.0-fold; yet this translates into a clinically important high
density lipoprotein raising effect (65).
is not expressed in preadipocytes and is turned on during
differentiation prior to the expression
LRP3 and other adipocyte
genes (66). In addition to LRP, many adipocyte proteins important in
triglyceride accumulation, such as lipoprotein lipase, fatty acid
transport protein-1, acyl-CoA synthase, CD36, and aP2 (67-70), all
contain at least one PPRE in their 5'-flanking sequences and are all
regulated by PPAR
. Adipocyte LRP functions in chylomicron remnant
cholesterol clearance both in vitro and in vivo
(24). We have recently demonstrated that LRP also plays a role in the
selective uptake of high density lipoprotein-CE by human adipocytes
(71). Thus, co-ordinate regulation of LRP and fatty acid transporters
may be a mechanism by which adipocytes can regulate cholesterol uptake
to match TG synthesis during differentiation and maturation of the
preadipocytes into fat cells.
,
, and
) have been identified. Within a given
species, the DNA-binding domains of the three PPARs are 80% identical
(slightly higher between PPAR
2 and PPAR
(72)). However, their
ligand-binding domains only share ~65% homology (73). It has been
demonstrated that PPAR
and PPAR
bind the same core DR-1 PPRE
(74). The distinct tissue-specific expression of the different PPARs as
well as their specific activation by ligands suggests a mechanism for
highly tissue-specific regulation of genes with a PPRE, including LRP.
PPAR
is predominantly expressed in liver, heart, kidney, intestinal
mucosa, and brown adipose tissue (33). These are all sites with high
fatty acid catabolism and peroxisomal metabolism. PPAR
is
ubiquitously expressed, whereas PPAR
is expressed mainly in adipose
tissue, skeletal muscle, heart, brain, vascular smooth muscle cells,
and monocyte/macrophages (31, 33, 75). PPAR
2 is relatively
adipose-specific, although in animal models of obesity, hepatic
expression of PPAR
2 has been documented (76). The transcriptional
activity of the PPAR subtypes is enhanced by a multitude of compounds.
Prostaglandin J2 is a natural ligand for PPAR
, whereas
thiazolidenediones (e.g. BRL49653 or rosiglitazone) are
synthetic ligands for PPAR
(36) and do not activate PPAR
. PPAR
ligands include 8(S)hydroxyeicosapentanoic acid, leukotriene B4, and
the synthetic fibrates. Long chain fatty acids are less specific
ligands recognizing all PPAR subtypes (33, 34). By administration of
selective PPAR ligands, it may be possible to regulate the expression
of LRP in a tissue-specific manner.
-mediated increase in hepatic LRP
expression may explain in part the triglyceride-lowering effects of
fibric acid derivatives. In ongoing studies, we are investigating
PPAR-mediated regulation of LRP expression and function in other cell
types, including hepatocytes and neuronal cells. The involvement of LRP
in a variety of important metabolic processes including amyloid
precursor protein processing,
-amyloid clearance, lipoprotein
metabolism, cellular remodeling, and protease complex clearance suggest
a possible therapeutic role for LRP up-regulation by PPAR ligands in a
number of disease states.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Dr. Steven Smith (Smith
Kline Beecham) for provision of rosiglitozone, Dr. Michael Saunders
(Glaxo Wellcome Inc.) for RXR, and Dr. Bruce Spiegelman for
PPAR
2. Thanks to Dr. Xiaohui Zha and members of the Lipoprotein
Group for technical advice and critical review of this manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by Heart and Stroke Foundation of Ontario Grant T-4631.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.
Supported by Ontario Graduate Scholarships in Science and
Technology and Ontario Graduate Scholarships.
§ The Wyeth Ayerst/Canadian Institutes of Health Research Chair in Cardiovascular Disease. To whom correspondence should be addressed: University of Ottawa, Rm. H441, 40 Ruskin St., Ottawa K1Y 4W7, Canada. Tel.: 613-761-5256; Fax: 613-761-5281; E-mail: rmcpherson@ottawaheart.ca.
Published, JBC Papers in Press, January 27, 2003, DOI 10.1074/jbc.M212989200
2 F. Benoist and R. McPherson, unpublished data.
3 F. Benoist and R. McPherson, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
LRP, low density
lipoprotein receptor-related protein;
PPAR, peroxisome
proliferator-activated receptor;
RXR, retinoid X receptor;
PPRE, peroxisome proliferator response element;
CS, fetal calf serum;
RT, reverse transcription;
2M,
2-macroglobulin;
2M*, activated
2M;
EMSA, electrophoretic mobility shift assay(s);
hACOX, human fatty acyl CoA oxidase;
PBS, phosphate-buffered saline;
RAP, receptor-associated protein;
BSA, bovine serum albumin;
HBSS, Hanks' balanced salt solution.
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