Modified methylenedioxyphenol analogs lower LDL cholesterol through induction of LDL receptor expression

Article abstract of DOI:10.1194/jlr.M022806

Although statin therapy is a cornerstone of current low density lipoprotein (LDL)-lowering strategies, there is a need for additional therapies to incrementally lower plasma LDL cholesterol. In this study, we investigated the effect of several methylenedioxyphenol derivatives in regulating LDL cholesterol through induction of LDL receptor (LDLR). INV-403, a modified methylenedioxyphenol derivative, increased LDLR mRNA and protein expression in HepG2 cells in a dose- and time-dependent fashion. These effects were apparent even under conditions of HMG-CoA reductase inhibition. Electrophoresis migration shift assays demonstrated that INV-403 activates SREBP2 but not SREBP1c, with immunoblot analysis showing an increased expression of the mature form of SREBP2. Knockdown of SREBP2 reduced the effect of INV-403 on LDLR expression. The activation of SREBP2 by INV-403 is partly mediated by Akt/GSK3β pathways through inhibition of phosphorylation-dependent degradation by ubiquitin-proteosome pathway. Treatment of C57Bl/6j mice with INV-403 for two weeks increased hepatic SREBP2 levels (mature form) and upregulated LDLR with concomitant lowering of plasma LDL levels. Transient expression of a LDLR promoter-reporter construct, a SRE-mutant LDLR promoter construct, and a SRE-only construct in HepG2 cells revealed an effect predominantly through a SRE-dependent mechanism. INV-403 lowered plasma LDL cholesterol levels through LDLR upregulation. These results indicate a role for small molecule approaches other than statins for lowering LDL cholesterol. Copyright

Full text of DOI:10.1194/jlr.M022806

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Modified methylenedioxyphenol analogs lower LDL
cholesterol through induction of LDL receptor expression
†
Zhekang Ying,* Rajagopal Desikan, Xiaohua Xu,* Andrei Maiseyeu,* Cuiqing Liu,*
1
,
Qinghua Sun,* Ouiliana Ziouzenkova,* Sampath Parthasarathy,* and Sanjay Rajagopalan *
†
Davis Heart and Lung Research Institute,* Ohio State University, Columbus, OH; and InVasc Therapeutics,
Tucker, GA
Abstract Although statin therapy is a cornerstone of cur-
rent low density lipoprotein (LDL)-lowering strategies, there
is a need for additional therapies to incrementally lower
plasma LDL cholesterol. In this study, we investigated the
effect of several methylenedioxyphenol derivatives in regu-
lating LDL cholesterol through induction of LDL receptor
Elevated levels of plasma low density lipoprotein (LDL)
cholesterol is a primary determinant of atherosclerosis (1, 2).
In humans, the majority of plasma cholesterol is carried by
the LDL fraction with cellular uptake of LDL being medi-
ated through LDL receptor (LDLR). The circulating level
of LDL is determined in large part by its rate of uptake
through the hepatic LDLR pathway, as evidenced by the
hypercholesterolemia in patients with defective LDLR or
ApoB-100 or elevated LDL levels in mice with a homozy-
gous deletion of the LDL receptor (3, 4).
Studies have shown that LDLR expression is finely attuned
to changes in intracellular cholesterol (5). A transcription
factor known as the sterol-responsive element binding pro-
tein 2 (SREBP2) plays a critical role in LDLR mRNA expres-
sion (6–8). After synthesis, SREBP2 forms a complex with the
SREBP cleavage-activating protein (SCAP) and is localized
in the endoplasmic reticulum (ER) as an inactive precursor
(pro-SREBP2). Sterol deficiency results in the release of
SREBP2/SCAP complex from ER and transport to the
Golgi, where pro-SREBP2 is processed further, allowing
the N-terminal fragment to enter the nucleus and upregu-
late transcription of LDLR (9). In addition to SREBP2, other
transcription factors may be involved in a context-dependent
fashion in regulating LDLR expression. For example, a muta-
tion at the Sp1 binding site (Ϫ49C>T) in the promoter of
LDLR gene is associated with familial hypercholesterolemia
(10). Upregulation of LDLR may thus represent an attractive
strategy to control plasma LDL cholesterol levels.
(
LDLR). INV-403, a modified methylenedioxyphenol deriv-
ative, increased LDLR mRNA and protein expression in
HepG2 cells in a dose- and time-dependent fashion. These
effects were apparent even under conditions of HMG-CoA
reductase inhibition. Electrophoresis migration shift assays
demonstrated that INV-403 activates SREBP2 but not
SREBP1c, with immunoblot analysis showing an increased
expression of the mature form of SREBP2. Knockdown of
SREBP2 reduced the effect of INV-403 on LDLR expres-
sion. The activation of SREBP2 by INV-403 is partly medi-
ated by Akt/GSK3␤ pathways through inhibition of
phosphorylation-dependent degradation by ubiquitin-prote-
osome pathway. Treatment of C57Bl/6j mice with INV-403
for two weeks increased hepatic SREBP2 levels (mature
form) and upregulated LDLR with concomitant lowering of
plasma LDL levels. Transient expression of a LDLR promoter-
reporter construct, a SRE-mutant LDLR promoter construct,
and a SRE-only construct in HepG2 cells revealed an effect
predominantly through a SRE-dependent mechanism.
INV-403 lowered plasma LDL cholesterol levels through
LDLR upregulation. These results indicate a role for small
molecule approaches other than statins for lowering LDL
cholesterol.—Ying, Z., R. Desikan, X. Xu, A. Maiseyeu, C. Liu,
Q. Sun, O. Ziouzenkova, S. Parthasarathy, and S. Rajagopalan.
Modified methylenedioxyphenol analogs lower LDL choles-
terol through induction of LDL receptor expression. J. Lipid
Res. 2012. 53: 879–887.
Various studies have shown that sesame oil can lower
LDL levels in animal models (11–13). The effects of sesame
oil have been attributed to both the fatty acid and non-
saponifiable components (11–13). Recent structure-activity
relationship analysis of berberine, a small molecule derived
from a plant, has demonstrated that the methylenedioxy-
phenyl group of berberine is critical for the induction of
Supplementary key words metabolism • low density lipoprotein •
lipoproteins/receptors • protein kinases
This work was supported by National Institutes of Health SBIR Grants
(
1R43HL103269-01 and 1R43HL103350-01) to Invasc and OSU and funds
from InVasc Therapeutics. Intellectual property in this grant belongs to OSU
and is licensed to InVasc Therapeutics. S. Rajagopalan is supported by John W.
Wolfe Professorship Funds. S. Parthasarathy is supported by National Institutes
of Health RO1 (AT-004106). R. Desikan is a full time employee of InVasc. Z.
Ying is supported by AHA Fellowship (11POST7640030). A. Maiseyeu is supported
by an AHA Fellowship Award (10POST4150090). The contents of this work are
solely the responsibility of the authors and do not necessarily represent the official
views of the National Institutes of Health or other granting agencies.
Abbreviations: LDLR, LDL receptor; MDP, methylenedioxyphenol;
PPAR, peroxisome proliferator-activated receptor; SRE, serum re-
sponse element; SREBP2, sterol-responsive element binding protein 2.
1
To whom correspondence should be addressed.
Manuscript received 2 December 2011 and in revised form 7 February 2012.
e-mail: sanjay.rajagopalan@osumc.edu
Published, JLR Papers in Press, February 21, 2012
DOI 10.1194/jlr.M022806
The online version of this article (available at http://www.jlr.org)
contains supplementary data in the form of four figures.
Copyright © 2012 by the American Society for Biochemistry and Molecular Biology, Inc.
This article is available online at http://www.jlr.org
Journal of Lipid Research Volume 53, 2012
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LDLR (14). We previously synthesized an analog of methyl-
enedioxyphenol (MDP), INV-403, and we demonstrated
that, in a model of LDLR deficiency, this small molecule
inhibited atherosclerosis, paralleled by a decrease in aortic
inflammation oxidative stress and improvement in endothe-
lial function without overt change in plasma lipid profile
and LDL (15). In the present study, we assess the effects of
INV-403 and related analogs on cholesterol metabolism
and LDLR expression, and we show that treatment with
INV-403 unexpectedly and dramatically reduced plasma
LDL, paralleled by an increase in the hepatic LDLR.
methods, respectively. The LDL cholesterol was measured by
Roche homogeneous LDL-C assay (Roche Diagnostics). The
HDL cholesterol was subsequently measured by precipitation
with phosphotungstic acid and MgCl2 (Roche Diagnostics).
VLDL cholesterol was calculated by subtraction of HDL and LDL
cholesterol from total cholesterol.
Western blot analysis
Tissue or cell lysates were prepared using radio-immunopre-
cipitation analysis (RIPA) buffer supplemented with protease and
phosphatase inhibitors. Protein samples were then separated by
8% (LDLR) or 10% (proteins other than LDLR) sodium dodecyl
sulfate polyacrylamide gel electrophoresis, and electro-blotted
onto polyvinylidene fluoride membranes. Target proteins were
detected by primary antibodies as follows: mouse anti-␤-actin
MATERIALS AND METHODS
Synthesis of INV-403
The final compound was obtained in two-step process.
In step I, an intermediate compound [2] is made by the simple
acetylationofmethylenedioxyphenol[1]. Acetylationwasachieved
using acetic anhydride in 75–80% chemical yield. The starting ma-
terial 3,4-methylenedioxyphenol (1.38 g, 0.01 mol) was dissolved
in 10% sodium hydroxide solution (15 ml), and to the cooled so-
lution, acetic anhydride (1.5 g, 0.015 mol) was added, with stirring
over 10 min. The reaction mixture was extracted with carbon tet-
rachloride. The extract was neutralized with sodium carbonate
solution and dried over magnesium sulfate. The solvent was re-
moved under reduced pressure and directly purified over column
chromatography, and purified product was used directly for next
step. The acetylated product serves as the starting point of many
other compounds, including nitro derivatives.
[
1]
[2]
In step II, the compound [2] from step I (1.8 g, 0.01 mol) was
dissolved in glacial acetic acid (15 ml) and was treated at room
temperature gradually with a solution of concentrated nitric acid
(
(
1.1 g, 0.012 mol; specific gravity 1.42) in glacial acetic acid
5 ml). The solution was stirred for 3 h and then poured into
cold water (50 ml). After 1 h, the pale-yellow crystalline precipi-
tate was separated by filtration, washed with water, and dried. 3,4-
methylenedioxy-6-nitrophenyl acetate (INV-403) was crystallized
from ethanol, yield 81%, melting point 104–105°. The purity of
obtained INV-403 was more than 99% as determined by nuclear
magnetic resonance spectroscopy (supplementary Fig. III).
[
2] [INV-403]
Animals
Fig. 1. INV-403 activates the LDLR promoter. (A) HepG2 cells
were transfected with pLDLR-luc plasmid. These cells were then
treated with the indicated compounds (10 ␮M) for varying times,
and then the luciferase activity in cell lysates was analyzed. n = 3.
The Institutional Animal Care and Use Committee (IACUC)
at Ohio State University approved the experimental animal pro-
tocols. Twelve male C57Bl/6j mice (8 weeks old) were obtained
from the Jackson Laboratory and allowed to acclimate for two
weeks. They were then intraperitoneally injected with vehicle
*P < 0.05 versus 0 h by one-way ANOVA. (B) HepG2 cells were trans-
fected with pLDLR-luc plasmid. After 24 h, these cells were treated
with the varying concentration of the indicated compounds for 4 h,
and then the luciferase activity in cell lysates was analyzed. n = 6;
(10% ethanol in phosphate buffered saline) or INV-403 (20 mg/
kg/day for two weeks).
*
P < 0.05 versus vehicle by one-way ANOVA. (C) HepG2 cells were
Plasma lipoprotein profile
transfected with pLDLR-luc plasmid. After 24 h, these cells were
pretreated with lovastatin (5 ␮M) or vehicle for 20 h, and treated
with INV-403 (10 ␮M) for 4 h, and then the luciferase activity in cell
Plasma lipoproteins were profiled by Cardiovascular Specialty
Laboratories Inc. (Atlanta, GA). Total cholesterol and triglyceride
concentrations were determined enzymatically with the CHOD-PAP
#
lysates was analyzed. n = 4; *P < 0.05 versus vehicle; P < 0.05 versus
(Roche Diagnostics) and lipase/GPO/PAP (Roche Diagnostics)
statin by one-way ANOVA.
8
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(
Sigma), mouse anti-LDLR (R and D Systems), mouse anti-
were visualized by a chemiluminescent reaction with streptavidin–
horseradish peroxidase and exposure of the blot to X-ray film
(Kodak). Specificity of the SREBP2 probe was confirmed in
assays in which unlabeled SREBP2 probe was added in excess as
a competitor and by the supershift of SREBP2-DNA complexes in
the presence of anti-SREBP2.
phosphoserine, rabbit anti-GSK3␤(s9), rabbit anti-GSK3␤(Y216),
mouse anti-GSK3␤, and anti-SREBP2 (Abcam). Secondary anti-
bodies conjugated with horseradish peroxidase and chemilumi-
nescence reagent (Amersham) were used to visualize the target
proteins. Densities of target protein bands were determined with
®
Quantity One 4.4.1 (Bio-Rad). The internal control, ␤-actin, was
used to normalize loading variations.
Real-time RT-PCR
Total RNA was extracted with TRI-Reagent® (Sigma, St. Louis,
MO) per the instruction. Real-time PCR was carried out using SYBR
Green as described previously (17). 2 [target genes relative to the
endogenous glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
control] were calculated as suggested by Schmittgen (18).
Electrophoretic mobility shift assay
⌬Ct
Nuclear proteins were extracted with NE-PER nuclear and
cytoplasmic extraction reagents (Pierce) from mouse livers or
HepG2 cells. The protein concentrations were determined by
TM
Bradford assay (Pierce). EMSAs were conducted with LightShift
LDL uptake assay
electrophoretic mobility shift assay (EMSA) kits (Pierce) per man-
ufacturer’s instructions. Briefly, 3 µg nuclear proteins/sample
were incubated with 15 pmol of biotinylated oligonucleotide
probe (5′-ATTTAGGTCCCTCCCCC CAACTTATGATTT-3′) (16)
in 25 mmol l-1 Hepes, pH 7.6, 100 mmol l-1 NaCl, 15% (v/v)
glycerol, 0.1% (v/v) NP-40, and 0.5 mmol l-1 PMSF in a final
volume of 20 µl. Incubations were conducted for 20 min at 18°C.
After incubation, the samples were electrophoresed for ap-
proximately 2 h at 100 V with 5% acrylamide nondenaturing
gels, and then transferred to nylon membranes via electroblot-
ting at 380 milliamperes for 30 min. The DNA-protein complexes
HepG2 cells were grown in DMEM containing 10% fetal bo-
vine serum and 1% penicillin/streptomycin. When the cells were
70–80% confluent, fresh medium without FBS was added con-
taining vehicle (1% DMSO) or different amounts of INV-403.
After incubation for 12 h, medium was removed and cells were
washed with PBS. Next, 25 ␮g/ml of BODIPY-LDL (Molecular
Probes) was added and incubated for 15 min. Cells were exten-
sively washed three times with PBS, and LDL uptake was quantified
on a fluorescence plate reader or visualized with confocal micros-
copy (following staining with DRAQ5 for nuclei). To quantify
Fig. 2. INV-403 increases LDLR expression and
function. (A) HepG2 cells were treated with INV-403
(10 ␮M) for the indicated time, and the LDLR mRNA
expression was then analyzed by real-time RT-PCR.
n = 3; *P < 0.05 versus 0 h by one-way ANOVA. (B)
HepG2 cells were treated with the indicated concen-
tration of INV-403 for 4 h, and the LDLR mRNA ex-
pression was then analyzed by real-time RT-PCR. n =
4
; *P < 0.05 versus vehicle by one-way ANOVA. (C, D).
HepG2 cells were treated with INV-403 (10 ␮M) for
the indicated time, and the LDLR protein expression
was then analyzed by Western blot. n = 3; *P < 0.05
versus 0 h by one-way ANOVA. (E, F). HepG2 cells
were treated with the indicated concentration of INV-
4
03 for 8 h, and the LDLR protein expression was
then analyzed by Western blot. n = 3; *P < 0.05 versus
h; one-way ANOVA. (G, H) HepG2 cells were
0
treated with vehicle or different concentrations of
INV-403 for 16 h. After washing with PBS, cells were
incubated with 25 ␮g/ml of BODIPY-LDL for 15 min.
Subsequent to extensively washing with PBS and
staining with DRAQ5 for nuclei, LDL uptake was visu-
alized with confocal microscopy (*P < 0.05 versus ve-
hicle by ANOVA; n = 3). The marked induction of
LDLR by INV-403 indicated that HepG2 cells are an
appropriate in vitro tool to define how INV-403 in-
creases LDLR.
INV-403 induces LDL receptor
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the confocal microscopy images, six images per slide were ran-
domly chosen. The fluorescent density of each image was ob-
tained and normalized by the density of DRAQ5 staining.
in HepG2 cells using a yeast UAS-TK system, indicating
that the effects of INV-403 on the LDLR promoter is spe-
cific and does not involve these pathways.
We next confirmed the effects of INV-403 on LDLR ex-
pression in HepG2 cells through assessment of LDLR
mRNA expression in response to INV-403 treatment [Fig. 2A,
B, half-maximal concentration (EC ): 20–800 nM]. Because
posttranslational regulation plays a critical role in LDLR
expression, we examined LDLR protein expression in
response to INV-403 treatment. Fig. 2C–F revealed that
consistent with the mRNA expression analysis, INV-403 time-
and dose-dependently increased LDLR protein expression
in HepG2 cells. The increased LDLR protein expression
was confirmed by the increased uptake of fluorescently la-
beled cholesterol in HepG2 cells (Fig. 2G, H).
As LDLR expression is exquisitely sensitive to choles-
terol levels and serum is the major source of exogenous
cholesterol for cultured cells, we assessed the effect of se-
rum on the induction of LDLR by INV-403. Fig. 3A reveals
that INV-403 activated the LDLR promoter, even in the
absence of serum, when it would be expected that there
Transfection and luciferase assay
The SREBP trans-activation activity luciferase reporter plasmid
(p6×SRE-Luc) was a gift from Dr. Andrew J. Brown (19). The lu-
50
ciferase reporter plasmids pLDLR-(wt)-Luc and pLDLR-(m)-Luc,
respectively, contained a 335 bp fragment of the ldlr promoter
with or without site-directed mutagenesis at SRE (ATCACCCCAC
changed to ATAACCCCAC) (8). They were purchased from
Addgene Inc. (Cambridge, MA). Transient transfection of semi-
confluent HepG2 cells in 60 mm dishes was performed using the
LipofectAMINE® reagent (Invitrogen, Carlsbad, CA) per manu-
facturer’s instructions. After 24 h, these cells were digested and
seeded into 96-well plates. After another 24 h, cells were treated
and then collected for luciferase activity assay. Each condition
was assayed in triplicate in every experiment, and each experi-
ment was repeated at least three times. Luciferase assays were
conducted using an assay kit from Sigma. Luciferase activities
were expressed as relative units after normalization to cotrans-
fected ␤-galactosidase (pcDNA3) activity using chlorophenol
red-␤-D-galactopyranoside substrate (Roche Diagnostics) as be-
fore (20). Results were combined from at least three indepen-
dent experiments.
Statistical analysis
Unless specifically mentioned, all results were expressed as
mean ± SEM. Probability values less than 0.05 were considered
significant. Student t-test or ANOVA were used for statistical anal-
ysis with GraphPad InStat 5 software (GraphPad Software Inc.,
San Diego, CA).
RESULTS
To investigate whether MDP or its analogs have the ca-
pacity to induce LDL receptor, we synthesized four mole-
cules beginning with MDP by way of introducing acetyl
group at phenolic OH and/or nitro group at the sixth po-
sition of MDP, and we compared their effects on LDLR
promoter–controlled luciferase expression. Whereas INV-
4
01 (I-acetoxymethylenedioxyphenol) did not activate the
LDLR promoter, INV-402 (6-nitromethylenedixoyphenol)
and INV-403 (O-acetoxynitromethylenedioxyphenol) time-
and dose-dependently induced luciferase activity in HepG2
cells, indicating that the introduction of the nitro group at
the sixth position may be essential for LDLR induction
(
Fig. 1A, B). There was no significant difference in activa-
tion of the LDLR promoter by INV-402 and INV-403. Given
that we previously observed that the acetylation of pheno-
lic OH increased the chemical stability (15), we used INV-
4
03 in all subsequent studies to investigate the mechanisms
of LDLR promoter activation. To determine the efficacy of
INV-403 on LDLR expression, we compared the effects of
INV-403 and a statin (lovastatin) on LDLR promoter activ-
ity. Fig. 1C shows that INV-403 and lovastatin had similar
effects on LDLR promoter activity, with the combination
increasing LDLR to levels higher than each compound indi-
vidually.Peroxisomeproliferator–activatedreceptors(PPAR)
are nuclear receptors that play an important role in lipid
metabolism (21). Supplementary Fig. I demonstrates that
Fig. 3. INV-403 increases LDLR expression whether or not there
is exogenous cholesterol. (A) After 24 h of transfection with pLDLR-
luc plasmid, HepG2 cells were cultured with the indicated concen-
tration of serum overnight, and then treated with INV-403 (10 ␮M)
for 4 h. The luciferase activity in cell lysates was analyzedand pre-
sented. n = 3; *P < 0.05 versus vehicle by one-way ANOVA. (B, C)
HepG2 cells were cultured in medium with the indicated concen-
tration of serum overnight and then treated with the indicated con-
centration of INV-403 for 8 h, and the LDLR protein expression
was analyzed by Western blot. N = 3; *P < 0.05 versus vehicle; one-way
ANOVA.
1
0 ␮M INV-403 did not activate PPAR␣, PPAR⌬, or PPAR␥
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Fig. 4. INV-403 activates SREBP2. (A–D) HepG2
cells were treated with INV-403 (10 ␮M) for the indi-
cated time (A and B) or the indicated concentration
of INV-403 for 1 h (C and D). Nuclear proteins were
extracted with NE-PER nuclear and cytoplasmic ex-
traction reagents (Pierce). The DNA binding activity
of SREBP2 was analyzed with a LightShift chemilumi-
nescent EMSA kit (Pierce). n = 4; *P < 0.05 versus
vehicle; one-way ANOVA. #, nonspecific band. (E–H)
HepG2 cells were stimulated with INV-403 (10 ␮M)
for the indicated time (E and F) or the indicated con-
centration of INV-403 for 1 h (G and F). The SREBP2
proteins in cell lysates, including the precursor and
mature forms, were analyzed by Western blot. n = 3;
*
P < 0.05 versus vehicle by one-way ANOVA.
would be maximal upregulation of LDLR expression. This
was demonstrated on analysis of LDLR protein expres-
sion under the same conditions (Fig. 3B, C). Although the
basal expression of LDLR was reduced, the relative in-
crease in LDLR with INV-403 was preserved.
SREBP2 is essential for the activation of the LDLR pro-
moter. INV-403 time- and dose-dependently increased
SREBP2 DNA binding activity in HepG2 cells, indicating
that SREBP2 plays a role in the induction of LDLR by INV-
To verify the role of SREBP2 in the induction of LDLR
by INV-403, we used the siRNA to knock down the expres-
sion of SREBP2 in HepG2 cells. SREBP2 siRNA markedly
reduced the effect of INV-403 on LDLR expression, indi-
cating that SREBP2 plays a role in the induction of LDLR
by INV-403 (Fig. 5A, B). However, even when SREBP2 was
knocked down with siRNA, a significant effect of INV-403
on LDLR expression was still observed. This was confirmed
by a reporter assay (Fig. 5C), suggesting that INV-403
may also increase LDLR through SREBP2-independent
mechanisms. To test whether INV-403 increases LDLR
through SREBP2-dependent and independent mecha-
nisms, we used various promoter-reporter constructs to as-
sess the effects of INV-403. Fig. 5D reveals that INV-403
increased luciferase expression (albeit at much lower lev-
els) under the control of the serum response element
(SRE) alone. Consistent with the important role of SRE
in the expression of LDLR, mutation of the SRE site
of the LDLR promoter dramatically decreased the lu-
ciferase expression. However, a small but significant residual
4
03 (Fig. 4A–D). In contrast, INV-403 did not affect the
activation of SREBP1c, the homolog of SREBP2 (supple-
mentary Fig. II). The activation of SREBP2 involves the
cleavage of SREBP2 precursor, with the mature form of
SREBP2 entering the nucleus to initiate transcription by
binding to consensus elements on the LDLR promoter.
To confirm the effect of INV-403 on SREBP2 activation,
we analyzed the maturation of SREBP2 in HepG2 cells
upon the treatment with INV-403. As shown in Fig. 4E–H,
INV-403 time- and dose-dependently increased mature
SREBP2 in HepG2 cells.
INV-403 induces LDL receptor
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Fig. 6. Akt/GSK3␤ mediates LDLR induction by INV-403. (A)
HepG2 cells were treated with INV-403 (10 ␮M) for the indicated
time, and the phosphorylation of Akt and GSK3␤ was analyzed by
Western blot. A representative blot from at least two independent
experiments with similar results is presented. (B) HepG2 cells were
treated with the indicated concentration of INV-403 for 4 h, and the
phosphorylation of Akt and GSK3␤ was analyzed by Western blot. A
representative blot from at least two independent experiments with
similar results is presented. (C) HepG2 cells were treated with INV-
403 (10 ␮M) or vehicle for 4 h, and anti-SREBP2 was used to precipi-
tate SREBP2 from cell lysates. The serine phosphorylation level and
total amount of SREBP2 were then analyzed by Western blot. (D)
HepG2 cells were transiently transfected with reporter constructs,
and following the indicated treatment, SREBP2 mature form and
GSK3␤ were analyzed by Western blot. A representative blot from at
least two independent experiments with similar results is presented.
(E) HepG2 cells were transiently transfected with reporter con-
structs, and following the indicated treatment, cells were lysed and
Fig. 5. INV-403 activates the LDLR promoter through SREBP2-
dependent and independent mechanisms. (A, B) After 24 h of
transfection with control or SREBP2 siRNA, HepG2 cells were
treated INV-403 (10 ␮M) for 1 h (SREBP2) or 8 h (LDLR). The
expression of LDLR and SREBP2 was then analyzed and presented.
n = 3; *P < 0.05 versus vehicle by one-way ANOVA. (C) After 24 h of
cotransfection with pLDLR-luc plasmid and control or SREBP2
siRNA, HepG2 cells were treated with INV-403 (10 ␮M) for 4 h.
The luciferase activity in cell lysates was analyzed and presented. n = 3;
*
P < 0.05 versus vehicle; one-way ANOVA. (D) HepG2 were tran-
siently transfected with pLDLR-(wt)-Luc or p6×SRE-Luc, and then
treated with INV-403 (10 ␮M) for 4 h. Cells were lysed and lu-
ciferase activity was analyzed. n = 3; *P < 0.05 versus 0 h or vehicle,
one-way ANOVA. (E) HepG2 were transiently transfected with
pLDLR-(wt)-Luc or pLDLR-(m)-Luc, and then treated with INV-
4
03 (10 ␮M) for 4 h. Cells were lysed and luciferase activity was ana-
#
lyzed. n = 3; *P < 0.05 versus 0 h or vehicle, one-way ANOVA.
luciferase activity were analyzed. n = 3; *P < 0.05 versus vehicle; P <
0.05 versus INV-403, one-way ANOVA. iAkt, inhibitor of Akt.
effect of INV-403 on luciferase expression remained
(
Fig. 5E).
reduced SREBP2 phosphorylation, suggesting that INV-403
may upregulate SREBP2 activity through inhibition of its
phosphorylation by GSK3␤. To further explore the signal-
ing events upstream from GSK-3␤, we investigated the effect
of INV-403 on Akt, which is well known to phosphorylate
GSK3␤ at Ser9, resulting in its inhibition. INV-403 time- and
concentration-dependently activated Akt, suggesting that the
effect of INV-403 on GSK3␤ activity may be mediated by
Akt (Fig. 6A, B). To confirm the role of Akt in the inhibi-
tion of GSK3␤ by INV-403, we analyzed the effects of
INV-403 on GSK3␤ activity in the presence of an Akt
inhibitor. Fig. 6D shows that Akt inhibitor markedly increased
INV-403–induced GSK3␤ phosphorylation at Ser9 and
The levels of SREBP2 mature form are regulated through
phosphorylation and subsequent ubiquitination. We inves-
tigated the role of GSK-3␤, a constitutively active serine/
threonine kinase that posttranslationally modifies a num-
ber of cell signaling proteins. INV-403 time- and concentra-
tion-dependently increased GSK3␤ phosphorylation at Ser9
(the inhibitory phosphorylation site) and decreased GSK3␤
phosphorylation at Tyr216 (the activating autophosphory-
laion site) (Fig. 6A, B). To verify the effect of INV-403 on
GSK3␤-mediated phosphorylation of SREBP2, we immuno-
precipitated SREBP2 and analyzed the phosphorylation of
SREBP2 in response to INV-403. Fig. 6C shows that INV-403
8
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Journal of Lipid Research Volume 53, 2012

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reduced phosphorylation at Tyr216, paralleled by a decrease
in mature SREBP2 level. Consistent with these observations,
the inhibition of Akt significantly reduced INV-403–induced
LDLR promoter activation, supporting that the Akt/GSK3␤
pathway plays a role in LDLR induction by INV-403.
lowered LDL cholesterol level in wild-type mice, it did not
Ϫ/Ϫ
decrease LDL cholesterol level in LDLR
mice, strongly
indicating that INV-403 lowers LDL cholesterol through
induction of LDLR.
Given the effects of INV-403 in promoting LDLR ex-
pression in HepG2 cells, we next investigated whether
INV-403 can induce LDLR in vivo. Treatment with INV-
DISCUSSION
4
03 significantly increased hepatic LDLR mRNA and pro-
The present study has several important findings regard-
ing small molecule approaches to regulate LDLR, potential
treatment strategies that may have important implications
for cholesterol lowering. The main findings of our study
are as follows: i) A novel small molecule analog of MDP
may increase LDLR expression in cultured HepG2 cells, even
under conditions of HMG-CoA reductase inhibition and
sterol excess; ii) the increase in LDLR occurs predominately
though SREBP2-dependent mechanisms; iii) Akt-mediated
GSK3␤ inhibition is involved in the activation of SREBP2
by INV-403; iv) increase in LDLR in liver in C57Bl/6 mice
following a two-week treatment were paralleled by lower
plasma LDL cholesterol levels and activation of SREBP2 in
tein levels in C57Bl/6j mice (Fig. 7A–C). EMSA revealed
that INV-403 significantly increased the DNA binding ac-
tivity and maturation of SREBP2 (Fig. 7D–G). Because he-
patic LDLR levels play an important role in determining
plasma LDL cholesterol, we next profiled plasma lipopro-
teins in INV-403–treated mice. Compared with vehicle,
INV-403 decreased plasma LDL cholesterol level by about
60%, whereas it did not significantly affect other lipoproteins
(
Table 1). To confirm that INV-403 lowers LDL choles-
terol level through LDLR-mediated pathways, we com-
pared its effect on LDL lowering in wild-type and LDLR
mice. Table 2 shows that, whereas INV-403 significantly
Ϫ/Ϫ
Fig. 7. INV-403 activates SREBP2 and increases LDLR in C57Bl/6j mice. Male C57Bl/6J mice (12 weeks
old; Jackson Laboratories) were intraperitoneally injected with INV-403 (20 mg/kg/day) or vehicle for 14
days. (A) Hepatic LDLR mRNA expression by real-time RT-PCR. (B, C) Hepatic LDLR mRNA expression by
Western blot. (D, E) Hepatic SREBP2 DNA binding activity by EMSA. #, nonspecific band. (F, G) Hepatic
SREBP2 expression. n = 6/group; *P < 0.05 versus control, Student t-test.
INV-403 induces LDL receptor
885

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TABLE 1. The lipoprotein effects of INV-403 in C57/Bl6 mice
with HepG2 cells did not reveal an inhibition of proteasome
activity by INV-403 in vitro (data not shown), indicating that
INV-403 may target upstream events.
Vehicle
INV-403
Total cholesterol (mg/dl)
Total glyceride (mg/dl)
HDL (mg/dl)
LDL (mg/dl)
VLDL (mg/dl)
70.7 ± 21.5
41.7 ± 16.2
41.4 ± 16
18.3 ± 8.9
9.7 ± 7.3
72.3 ± 9.9
41.4 ± 11.7
52.4 ± 5.2
7.3 ± 0.9
14.7 ± 6.4
GSK3␤ is an ubiquitously expressed Ser/Thr kinase in-
volved in a variety of cellular processes, ranging from glyco-
gen metabolism and insulin signaling to cellular processes,
such as proliferation. It has been shown to phosphorylate
SREBP2 and target it to proteasome through ubiquitina-
tion-dependent mechanism (24). Our data demonstrate
that INV-403 activates GSK3␤ with a time course and a
dose-dependent response similar to that of SREBP2 activa-
tion, indicating that it may play a role in the SREBP2-medi-
ated LDLR induction by INV-403. Interestingly, GSK3␤
appears to phosphorylate SREBP2 mature form only (Fig.
a
Mice were intraperitoneally injected with INV-403 (20 mg/kg/day)
for 14 days. Plasma were then collected and lipoproteins were profiled.
a
P < 0.05 versus vehicle; Student t-test. n = 6/group.
liver; and v) LDL-lowering effects were not demonstrable
in the absence of LDLR.
The results in this study may provide additional targets
for regulation of plasma LDL cholesterol. LDLR regula-
tion by SREBP2 represents a potentially important thera-
peutic target in regulation of plasma LDL levels given its
central role in regulating cellular and plasma cholesterol
levels (6, 22, 23). In the present study, nitro derivatives of
a small molecule derivative of sesame oil lignans are shown
to have the potential to activate the LDLR promoter even
under conditions where cholesterol levels within the cell
are low (low sterol conditions) and during inhibition of
HMG-CoA reductase by a statin. Indeed, the results indi-
cate that the effects of INV-403 on LDLR promoter activity
may be additive to that of statin therapy. Thus, there may
be a potential to regulate LDLR expression overriding an
endogenous feed-back loop that is remarkably efficient
and sensitive to even small changes in intracellular choles-
terol concentration.
6C). This is consistent with a previous study showing that
the phosphorylation of SREBP2 by GSK3␤ is DNA-bind-
ing–dependent (24). The inhibition of Akt decreased
LDLR induction by INV-403 as shown by reporter assays,
offering evidence that Akt/GSK3␤ pathway may mediate
the LDL-lowering effect of INV-403. However, the extent
to which Akt/GSK3␤ pathway contributes to the LDL-low-
ering effect of INV-403 remains to be determined.
Although SREBP-2 through binding of the SRE undoubt-
edly plays the predominant role in INV-403 effects, our
data also suggest a role for SRE/SREBP-2–independent ef-
fects, albeit to a much lower level compared with that
through the SREBP2-dependent pathway (Fig. 5D). This is
consistent with a growing number of studies showing that
transcription factors other than SREBPs and cis-elements
other than SRE are involved in the LDLR transcription
regulation. For example, both Sp1 and estrogen receptor ␣
Our data indicate that increased LDLR expression INV-
03 is at least partly mediated by SREBP2 mediated activa-
4
(
(
ER␣) are required for the LDLR induction by estrogen
25). Inhibition of PPAR␥ coactivator-1␣ (PGC-1␣) can
tion of the LDLR promoter. This is based on the following
observations: i) INV-403 increased LDLR promoter activity;
ii) INV-403 Increased mature SREBP2 levels and upregu-
lated its DNA binding activity; iii) LDLR promoter activity
in response to INV-403 was substantially attenuated by a
truncated SRE-only construct; and iv) loss of function of
SREBP2 decreased the LDLR induction by INV-403. Nota-
bly, the increase in SREBP2 mature form was not propor-
tional to the decrease in SREBP2 precursor (Figs. 4E, G and
also contribute to LDLR induction by estrogen (26). The
induction of LDLR by berberine appears to require c-jun
(14). CCAAT–enhancer-binding protein (C/EBP) is nec-
essary for the induction of LDLR by oncostatin M (27–29).
Notably, INV-403 activated the LDLR promoter far more
efficiently than a truncated SRE-only promoter, indicating
that there may be an important contribution of other cis-
acting elements. However, because INV-403 activated the
SRE-mutated LDLR promoter to a much lower level, it re-
mains to be determined whether the SREBP2-independent
mechanism is pharmacologically meaningful.
5
A), indicating that the stabilization of mature SREBP2 may
be involved in the increase of SREBP2 activity in response to
INV-403 treatment. The Proteasome-Glo™ cell-based assays
The present study also provides evidence that the effects
of INV-403 are selective for LDL with no discernible ef-
fects on other lipoprotein fractions. We previously showed
that INV-403 had no LDL-lowering effects in LDLR-defi-
cient WHHL rabbits (15). Consistent with this data, our
current study, while demonstrating an effect of LDL lower-
TABLE 2. The differential lipoprotein effects of INV-403 in C57/Bl6
Ϫ/Ϫ
and LDLR
mice
Ϫ/Ϫ
Wt
LDLR
Vehicle
INV-403
Vehicle
INV-403
Total cholesterol 94.6 ± 11.8 102.6 ± 13.7 337.8 ± 44.8 389.4 ± 55.1
mg/dl)
Total glyceride
mg/dl)
ing in wild-type mice, was unable to discern an effect on
(
Ϫ/Ϫ
LDL lowering in LDLR
mice, indicating that LDLR
67.4 ± 22.8 53.7 ± 15.1 142.1 ± 29.4 125.5 ± 20
(
may be essential for the LDL-lowering action of INV-403.
There were no effects on plasma triglycerides, consistent
HDL (mg/dl)
LDL (mg/dl)
60.6 ± 7.9
25.4 ± 8.6
71.6 ± 6.7
14.6 ± 1.1 141.8 ± 26.6
81.9 ± 15
92.6 ± 8.7
167 ± 24.6
a
with an exclusive effect on LDL pathways as noted with the
VLDL (mg/dl) 10.8 ± 3
13.4 ± 5.5 114.1 ± 13.4 121.8 ± 29
Ϫ/Ϫ
phenotype of PCSK9
mice (30). The effects of this mol-
Mice were intraperitoneally injected with INV-403 (20 mg/kg/
day) for 14 days. Plasma samples were then collected and lipoproteins
ecule appear to be specific for SREBP2 with no evidence
of activation of SREBP1c (supplementary Fig. II). The ab-
sence of effects on triglyceride levels is again reassuring
were profiled.
a
P < 0.05 versus vehicle; Student t-test. n = 6/group.
8
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.
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and supports the lack of effect on SREBP-1c. Further,
there is no evidence that this activates other pathways,
such as PPAR␣, PPAR␥, or PPAR⌬ (supplementary Fig. I).
These results are different from those noted with ber-
berine, which has also been reported to lower LDL through
upregulation of LDLR but which was accompanied by sig-
nificant decrease in plasma TG level in humans (31). This
may reflect LDLR-independent effects of berberine.
Previous studies have shown that the methylenedioxy-
phenyl group is essential for the induction of LDLR by
berberine (14). However, our data demonstrate that the
MDP in and of itself cannot induce LDLR, indicating that
it may be essential but not sufficient for LDLR induction.
Our study has multiple important limitations that must
be acknowledged. First, we have not provided LDL-lowering
effects in a model of dyslipidemia with an intact LDLR
11. Reena, M. B., L. R. Gowda, and B. R. Lokesh. 2011. Enhanced hy-
pocholesterolemic effects of interesterified oils are mediated by up-
regulating LDL receptor and cholesterol 7-alpha- hydroxylase gene
expression in rats. J. Nutr. 141: 24–30.
12. Reena, M. B., and B. R. Lokesh. 2007. Hypolipidemic effect of oils
with balanced amounts of fatty acids obtained by blending and
interesterification of coconut oil with rice bran oil or sesame oil.
J. Agric. Food Chem. 55: 10461–10469.
13. Bhaskaran, S., N. Santanam, M. Penumetcha, and S. Parthasarathy.
2
006. Inhibition of atherosclerosis in low-density lipoprotein recep-
tor-negative mice by sesame oil. J. Med. Food. 9: 487–490.
4. Lee, S., H. J. Lim, J. H. Park, K. S. Lee, Y. Jang, and H. Y. Park.
2007. Berberine-induced LDLR up-regulation involves JNK path-
way. Biochem. Biophys. Res. Commun. 362: 853–857.
5. Ying, Z., N. Kherada, T. Kampfrath, G. Mihai, O. Simonetti, R.
Desikan, K. Selvendiran, Q. Sun, O. Ziouzenkova, S. Parthasarathy,
et al. 2011. A modified sesamol derivative inhibits progression of
atherosclerosis. Arterioscler. Thromb. Vasc. Biol. 31: 536–542.
1
1
1
6. Seo, Y. K., B. Zhu, T. I. Jeon, and T. F. Osborne. 2009. Regulation
of steroid 5-alpha reductase type 2 (Srd5a2) by sterol regulatory
element binding proteins and statin. Exp. Cell Res. 315: 3133–3139.
(
such as a hamster model of dyslipidemia) and this may
17. Ying, Z., P. Yue, X. Xu, M. Zhong, Q. Sun, M. Mikolaj, A. Wang,
R. D. Brook, L. C. Chen, and S. Rajagopalan. 2009. Air pollution
and cardiac remodeling: a role for RhoA/Rho-kinase. Am. J. Physiol.
Heart Circ. Physiol. 296: H1540–H1550.
18. Schmittgen, T. D., and B. A. Zakrajsek. 2000. Effect of experimen-
tal treatment on housekeeping gene expression: validation by real-
time, quantitative RT-PCR. J. Biochem. Biophys. Methods. 46: 69–81.
require additional experimentation. We have not provided
evidence that the LDL lowering results in reduction in
atherosclerosis. Finally, we have not provided definitive
evidence of the precise mechanism by which this drug
posttranslationally regulates SREBP2 that is distinguish-
able from other classic triggers of SREBP2 activation, such
as intracellular sterol deficiency, which is well known to
activate not only LDLR but also HMG-COA reductase.
Our findings suggest that small molecule strategies that
regulate LDLR and SREBP2 represent a promising area of
investigation.
1
9. Wong, J., C. M. Quinn, and A. J. Brown. 2006. SREBP-2 positively
regulates transcription of the cholesterol efflux gene, ABCA1, by
generating oxysterol ligands for LXR. Biochem. J. 400: 485–491.
0. Ziouzenkova, O., S. Perrey, L. Asatryan, J. Hwang, K. L. MacNaul,
D. E. Moller, D. J. Rader, A. Sevanian, R. Zechner, G. Hoefler,
et al. 2003. Lipolysis of triglyceride-rich lipoproteins generates
PPAR ligands: evidence for an antiinflammatory role for lipopro-
tein lipase. Proc. Natl. Acad. Sci. USA. 100: 2730–2735.
2
2
1. Rosenson, R. S. 2007. Effects of peroxisome proliferator-activated
receptors on lipoprotein metabolism and glucose control in type
2
diabetes mellitus. Am. J. Cardiol. 99: 96B–104B.
REFERENCES
2
2. Rideout, T. C., Z. Yuan, M. Bakovic, Q. Liu, R. K. Li, Y. Mine, and
M. Z. Fan. 2007. Guar gum consumption increases hepatic nuclear
SREBP2 and LDL receptor expression in pigs fed an atherogenic
diet. J. Nutr. 137: 568–572.
23. Eberlé, D., B. Hegarty, P. Bossard, P. Ferré, and F. Foufelle. 2004.
SREBP transcription factors: master regulators of lipid homeosta-
sis. Biochimie. 86: 839–848.
24. Du, X., I. Kristiana, J. Wong, and A. J. Brown. 2006. Involvement of
Akt in ER-to-Golgi transport of SCAP/SREBP: a link between a key
cell proliferative pathway and membrane synthesis. Mol. Biol. Cell.
17: 2735–2745.
25. Li, C., M. R. Briggs, T. E. Ahlborn, F. B. Kraemer, and J. Liu. 2001.
Requirement of Sp1 and estrogen receptor alpha interaction in 17beta-
estradiol-mediated transcriptional activation of the low density lipo-
protein receptor gene expression. Endocrinology. 142: 1546–1553.
26. Jeong, J. H., S. Cho, and Y. K. Pak. 2009. Sterol-independent repres-
sion of low density lipoprotein receptor promoter by peroxisome
proliferator activated receptor gamma coactivator-1alpha (PGC-
1alpha). Exp. Mol. Med. 41: 406–416.
1
2
. Lusis, A. J. 2000. Atherosclerosis. Nature. 407: 233–241.
. Rader, D. J., and A. Daugherty. 2008. Translating molecular discov-
eries into new therapies for atherosclerosis. Nature. 451: 904–913.
. Brugger, D., H. Schuster, and N. Zollner. 1996. Familial hypercho-
lesterolemia and familial defective apolipoprotein B-100: compari-
son of the phenotypic expression In 116 cases. Eur. J. Med. Res. 1:
3
4
3
83–386.
. Ishibashi, S., M. S. Brown, J. L. Goldstein, R. D. Gerard, R. E.
Hammer, and J. Herz. 1993. Hypercholesterolemia in low density
lipoprotein receptor knockout mice and its reversal by adenovirus-
mediated gene delivery. J. Clin. Invest. 92: 883–893.
. Hussain, M. M. 2001. Structural, biochemical and signaling prop-
erties of the low-density lipoprotein receptor gene family. Front.
Biosci. 6: D417–D428.
. Horton, J. D., I. Shimomura, M. S. Brown, R. E. Hammer, J. L.
Goldstein, and H. Shimano. 1998. Activation of cholesterol synthe-
sis in preference to fatty acid synthesis in liver and adipose tissue of
transgenic mice overproducing sterol regulatory element-binding
protein-2. J. Clin. Invest. 101: 2331–2339.
. Hua, X., C. Yokoyama, J. Wu, M. R. Briggs, M. S. Brown, J. L.
Goldstein, and X. Wang. 1993. SREBP-2, a second basic-helix-loop-
helix-leucine zipper protein that stimulates transcription by bind-
ing to a sterol regulatory element. Proc. Natl. Acad. Sci. USA. 90:
5
6
27. Zhang, F., M. Lin, P. Abidi, G. Thiel, and J. Liu. 2003. Specific in-
teraction of Egr1 and c/EBPbeta leads to the transcriptional activa-
tion of the human low density lipoprotein receptor gene. J. Biol.
Chem. 278: 44246–44254.
7
8
9
28. Zhang, F., T. E. Ahlborn, C. Li, F. B. Kraemer, and J. Liu. 2002.
Identification of Egr1 as the oncostatin M-induced transcription
activator that binds to sterol-independent regulatory element of
human LDL receptor promoter. J. Lipid Res. 43: 1477–1485.
29. Zhou, Y., F. Zhang, P. Abidi, M. Lin, G. Thiel, and J. Liu. 2006. Blockage
of oncostatin M-induced LDL receptor gene transcription by a domi-
nant-negative mutant of C/EBPbeta. Biochem. J. 397: 101–108.
30. Rashid, S., D. E. Curtis, R. Garuti, N. N. Anderson, Y. Bashmakov,
Y. K. Ho, R. E. Hammer, Y. A. Moon, and J. D. Horton. 2005.
Decreased plasma cholesterol and hypersensitivity to statins in
mice lacking Pcsk9. Proc. Natl. Acad. Sci. USA. 102: 5374–5379.
31. Kong, W., J. Wei, P. Abidi, M. Lin, S. Inaba, C. Li, Y. Wang, Z. Wang,
S. Si, H. Pan, et al. 2004. Berberine is a novel cholesterol-lowering
drug working through a unique mechanism distinct from statins.
Nat. Med. 10: 1344–1351.
1
1603–11607.
. Castoreno, A. B., Y. Wang, W. Stockinger, L. A. Jarzylo, H. Du, J.
C. Pagnon, E. C. Shieh, and A. Nohturfft. 2005. Transcriptional
regulation of phagocytosis-induced membrane biogenesis by sterol
regulatory element binding proteins. Proc. Natl. Acad. Sci. USA. 102:
1
3129–13134.
. Brown, M. S., and J. L. Goldstein. 1997. The SREBP pathway: reg-
ulation of cholesterol metabolism by proteolysis of a membrane-
bound transcription factor. Cell. 89: 331–340.
0. Mozas, P., R. Galetto, M. Albajar, E. Ros, M. Pocovi, and J. C.
Rodriguez-Rey. 2002. A mutation (-49C>T) in the promoter of the
low density lipoprotein receptor gene associated with familial hy-
percholesterolemia. J. Lipid Res. 43: 13–18.
1
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