Received for publication, November 14, 2002, and in revised form, December 26, 2002
Fibrates are normolipidemic drugs used in
atherogenic dyslipidemia because of their ability to raise high density
lipoprotein (HDL) and decrease triglyceride levels. They exert multiple
effects on lipid metabolism by activating the peroxisome
proliferator-activated receptor-
(PPAR-
), which controls the
transcriptional regulation of genes involved in hepatic fatty acid,
cholesterol, and lipoprotein metabolism. The hepatic expression
of the scavenger receptor class B type I (SR-BI) plays a critical role
in lipoprotein metabolism, mainly due to its ability to mediate
selective cholesterol uptake. Because fibrates and PPAR-
agonists
up-regulate SR-BI expression in human and murine macrophages, we tested
whether fibrates raised a similar regulatory response on hepatic SR-BI
expression in mice. Surprisingly, fibrate treatment suppressed SR-BI
protein expression in the liver without changing steady state SR-BI
mRNA levels. Decreased hepatic SR-BI protein expression correlated
with enlarged HDL particle size. This effect was concomitant with
down-regulation of CLAMP, a putative SR-BI-stabilizing protein found in
the hepatic plasma membrane, which was also not associated to changes
in CLAMP mRNA levels. The post-transcriptional regulatory effect of
fibrates over hepatic SR-BI protein levels was dependent on PPAR-
expression, because it was absent in PPAR-
-deficient mice. Restoring
hepatic SR-BI expression in fibrate-treated mice by recombinant
adenoviral gene transfer abolished fibrate-mediated HDL particle size
enlargement. This study describes a novel effect of fibrates on hepatic
SR-BI expression providing an alternative mechanism by which this drug family modulates HDL metabolism in vivo.
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INTRODUCTION |
The long standing epidemiological evidence correlating increased
plasma high density lipoprotein cholesterol
(HDL-C)1 levels with
protection against atherosclerotic cardiovascular disease (reviewed in
Refs. 1 and 2) has attracted significant attention toward the
regulation of HDL-C homeostasis and the development of drugs directed
to beneficially modulate HDL-C metabolism. Plasma levels of HDL-C are
determined by a complex network of interactions between lipids
(e.g. fatty acids and cholesterol), circulating apolipoproteins (e.g. apoA-I and apoA-II), and lipid
transfer proteins (e.g. phospholipid transfer protein (PLTP)
and cholesteryl ester transfer protein), extracellular lipases
(e.g. hepatic lipase), cell surface receptors, and plasma
membrane lipid transporters (e.g. ATP-binding cassette
transporter A1). The scavenger receptor class B type I (SR-BI), a
cell surface HDL receptor capable of mediating selective HDL
cholesterol uptake (reviewed in Refs. 3-5), plays a distinct role in
HDL metabolism in mice by modulating plasma HDL-C levels and HDL
particle size and composition (6). Manipulations of hepatic SR-BI
expression levels by adenoviral gene transfer (7), transgenesis (8, 9),
and targeted gene ablation (6) have profound influence on HDL-C levels
and its availability for biliary cholesterol secretion in mice.
Furthermore, additional studies have established an inverse correlation
between murine hepatic SR-BI expression and atherosclerotic ischemic
heart disease (10-13). Taken together, these studies strongly suggest that pharmacological agents that modulate SR-BI expression and/or activity in the liver may have significant impact on HDL metabolism, reverse cholesterol transport, and atherosclerotic cardiovascular disease.
Fibrates are commonly used normolipidemic drugs that efficiently
decrease triglycerides and raise HDL levels in humans (14), resulting
in an overall reduction of coronary heart disease risk and events
(15-17). Several studies in animal models and cultured cells have
established that the normolipidemic effects of fibrates occur mainly
through transcriptional modulation of target genes involved in fatty
acid, triglyceride, and cholesterol metabolism and also in lipoprotein
formation and remodeling (18). This fibrate-mediated transcriptional
regulation is caused by binding and activation of a specific nuclear
receptor termed peroxisome proliferator-activated receptor-
(PPAR-
) (reviewed in Refs. 19 and 20). Indeed, fibrate-induced
effects on gene transcription depend on the presence of functional
PPAR-
-response elements in the promoter region of target genes (14,
19). Furthermore, PPAR-
knockout mice lack the above-mentioned lipid
metabolism-related responses associated with fibrate treatment (21),
indicating the essential role of this receptor in mediating fibrate action.
Whereas the role of fibrates in the regulation of plasma HDL-C levels
through changes in expression of plasma apoA-I (22, 23), apoA-II (24),
PLTP (25), lipoprotein lipase (26), and macrophage ATP-binding cassette
transporter A1 transporter (27) has been studied extensively, much less
is known about fibrate-dependent regulation of SR-BI.
Recently, Chinetti et al. (28) have shown that PPAR-
activation increased SR-BI protein levels in cultured human monocytes
as well as in fully differentiated macrophages. In addition,
fenofibrate treatment elevated SR-BI protein content in macrophages of
atherosclerotic lesions in apolipoprotein E (apoE) knockout mice (28).
These findings suggested that fibrates might modulate HDL metabolism by
increasing SR-BI expression in peripheral tissues.
Despite the importance of hepatic SR-BI expression for HDL metabolism
and the pleiotropic effects of fibrates on HDL metabolism-related proteins in the liver, the consequences of fibrate administration on
hepatic SR-BI expression have not been reported. In this study, we
determined hepatic SR-BI protein and mRNA levels in fibrate-treated mice, and we correlated these findings with plasma lipoprotein cholesterol profiles. We also examined the effect of fibrates on
hepatic protein and mRNA levels of CLAMP, a putative
SR-BI-stabilizing protein expressed in the liver (29). The PPAR-
dependence of changes in hepatic SR-BI protein expression induced by
fibrates was evaluated in fibrate-treated PPAR-
knockout mice.
Finally, we tested the physiological relevance of the fibrate-induced
SR-BI deficiency in the liver by restoring hepatic SR-BI expression through recombinant adenoviral gene transfer in fibrate-treated mice.
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EXPERIMENTAL PROCEDURES |
Animals and Fibrate Treatment--
Control male C57BL/6 mice
(2-3 months old), originally obtained from The Jackson Laboratory (Bar
Harbor, ME), were housed in a temperature- and humidity-controlled room
with reverse light cycling and fed a low cholesterol-containing chow
diet (ProLab RMH 3000; PMI Feeds, St. Louis, MO), with food and water
available ad libitum. For plasma cholesterol and hepatic
SR-BI and CLAMP expression studies, mice were switched to diets
containing 0.2% ciprofibrate (Sanofi, Gentilly, France) or 0.2%
fenofibrate (Sigma) for 7 days. For dose- and time-response studies,
mice were fed with diets supplemented with ciprofibrate at the
indicated doses and for the indicated times. To test the PPAR-
dependence of SR-BI regulation by fibrates, control and PPAR-
knockout mice (30) were fed with chow diet supplemented with 0.2%
fenofibrate for 14 days.
Recombinant Adenovirus Infection in Fibrate-treated Mice--
At
day 0, control C57BL/6 mice (2-3 months old) were separated in six
groups of four animals each and were switched to experimental diets as
follows: one group was kept in control diet, three groups were switched
to 0.2% ciprofibrate diet, and two groups were switched to 0.2%
fenofibrate diet. At day 4, one group of mice treated with either
ciprofibrate or fenofibrate were injected via the femoral vein with
5 × 1010 particles of murine sr-bi
recombinant adenovirus (Ad.mSR-BI, see Ref. 7). Another group of
ciprofibrate-treated mice was injected with 1 × 1011
particles of lacZ recombinant adenovirus (Ad.lacZ,
see Ref. 7) as control for the adenoviral infection. Regardless of the
use of adenoviruses, all groups were maintained until day 9 in their respective fibrate treatments.
Hepatocyte Culture and Fibrate Treatment--
Rat hepatocytes
were isolated and cultured as described previously (31). Briefly,
hepatocytes were prepared with collagenase perfusion from liver of male
Sprague-Dawley rats. Isolated hepatocytes were seeded (105
cells/well) in 6-well plates for 24 h in Dulbecco's modified Eagle's medium containing 10% fetal calf serum and 1% glutamine without or with supplementation with 100 µM fenofibric
acid. Cells were lysed with 0.2% SDS, 2% Nonidet P-40, 2 mM 2-mercaptoethanol, and protease inhibitors (ICN
Biomedicals mixture kit) in 0.1 M phosphate buffer, and
post-nuclear extracts were prepared by centrifugation at 10,000 × g for 15 min at 4 °C.
Blood and Liver Sampling and Processing--
After fibrate
treatment with or without associated adenoviral infections, mice were
anesthetized with pentobarbital (4.5 mg/100 g body weight) by
intraperitoneal injection. The abdomen was opened and blood was removed
by puncture of the inferior vena cava with a heparinized syringe; mice
were euthanized, and livers were removed. Plasma was separated by low
speed centrifugation for 10 min at 4 °C and kept at
20 °C,
whereas liver was stored at
70 °C for further biochemical
analyses. Total membrane extracts (postnuclear 100,000 × g membrane pellets) from individual liver samples were prepared as described (32).
Plasma Lipoprotein Analysis--
Size fractionation of plasma
lipoproteins was performed by fast performance liquid chromatography
(FPLC) of pooled plasma samples, and total cholesterol content on each
fraction was assayed enzymatically (33). Results are expressed as
micrograms of total cholesterol per FPLC fraction.
Immunoblotting Analysis--
Hepatocyte post-nuclear lysates (60 µg of protein/sample) or total liver membranes (40-50 µg of
protein/sample) were size-fractionated by 10% SDS-PAGE and
immunoblotted on nitrocellulose with either a polyclonal antipeptide
antibody against murine SR-BI protein (34), a polyclonal antibody
generated against the entire SR-BI protein (35), or a monoclonal
antibody against recombinant CLAMP (29). Polyclonal anti-
-COP (36)
or anti-actin (Santa Cruz Biotechnology, Santa Cruz, CA) antibodies
were used as protein loading control. Antibody binding to protein
samples was visualized by the enhanced chemiluminescence procedure
(Amersham Biosciences) and quantified with a Macintosh Color One
scanner (Apple, Cupertino, CA) and NIH imaging software version 1.6. SR-BI and CLAMP expression levels were normalized to the signal of
-COP or actin proteins detected on the same nitrocellulose membrane.
Immunofluorescence Analysis--
Mice were anesthetized by
intraperitoneal injection of pentobarbital; the abdominal cavity was
opened, and livers were perfused with cold phosphate-buffered saline
(PBS) through the portal vein. Livers were then excised and
frozen under 2-methylbutane in liquid nitrogen. Cryosections
(4-5 µm) of liver tissue were fixed in 7% formaldehyde solution in
PBS for 15 min, rinsed 3 times in PBS, and permeabilized with 0.1%
Triton X-100 for 15 min, blocked overnight in 10% goat serum in PBS,
and incubated for 2 h at room temperature with a polyclonal
antibody generated against the entire SR-BI protein (35) (dilution
1:80). As secondary antibody, fluorescein isothiocyanate
(FITC)-conjugated goat anti-rabbit IgG (Kirkegaard & Perry Laboratories
Inc., Gaithersburg, MD) (dilution 1:100) was used. Negative controls
were performed by omitting the primary antibody. After washing in PBS,
samples were mounted with coverslips using Fluoromount-G (Electron
Microscopy Sciences, Fort Washington, PA). Stained sections were
examined by immunofluorescence microscopy.
RNA Analysis--
Total RNA was prepared from mouse liver by the
acid guanidinium thiocyanate-phenol chloroform method (37). RNA samples
(20 µg/lane) were size fractionated by formaldehyde-agarose gel
electrophoresis and transferred to nylon. cDNA probes for SR-BI,
CLAMP, and 18 S rRNA were prepared by standard reverse
transcriptase-PCR using primers designed on cDNA sequences
available through GenBankTM data bases. All probes were
labeled by the random primer method (Promega, Madison, WI) and used for
Northern blot hybridization as described previously (38).
Quantification was performed by PhosphorImaging with the GS-525
Molecular Image System (Bio-Rad). The relative levels of SR-BI and
CLAMP mRNAs were determined by normalizing in the same filter to
the levels of 18 S rRNA.
Statistical Analysis--
Results of hepatic protein and
mRNA levels are expressed as fold change relative to the control
group. The statistical significance of the differences between the
means of the experimental groups was tested by the Student's
t test or variance analysis. A difference was considered
statistically significant when p < 0.05.
 |
RESULTS |
Hepatic SR-BI Protein Levels in Fibrate-treated Mice--
In a
previous study, Chinetti et al. (28) have shown that
treatment with different PPAR-
agonists increased SR-BI protein mass
in human macrophages and in atherosclerotic lesions of apoE-deficient mice. Here we tested two commonly used fibrates, ciprofibrate and
fenofibrate, for the ability to modulate SR-BI protein levels in the
murine liver. Treatment of mice with either 0.2% ciprofibrate or 0.2%
fenofibrate drastically decreased SR-BI protein to undetectable levels
when analyzed by immunoblotting in total liver membranes (Fig.
1A). The reduction of hepatic
SR-BI protein levels by fibrates was dose- and
time-dependent (Fig. 1, B and C),
reaching its maximal effect at 0.2% ciprofibrate and after 48 h
of treatment with 0.02% ciprofibrate.

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Fig. 1.
Hepatic SR-BI protein expression analysis by
immunoblotting and immunofluorescence in control and fibrate-treated
mice. Wild-type C57BL/6 mice were fed with standard chow diet
without (control) and with supplementation with fibrates as follows:
A, 0.2% ciprofibrate (CF) or 0.2% fenofibrate
(FF) for 7 days; B, ciprofibrate at increasing
doses for 7 days; C, 0.02% ciprofibrate for increasing
times; D, 0.2% ciprofibrate for 7 days. Liver samples were
removed and processed for immunoblotting and immunofluorescence as
described under "Experimental Procedures." Primary antibody binding
was detected by appropriate secondary antibodies for chemiluminescence
(A-C) or fluorescence microscopy (D). SR-BI
protein levels are expressed as fold change relative to chow-fed mice
after correction for -COP level (n = 4; *,
p < 0.01). The results of this figure are
representative of at least 3 independent experiments.
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To confirm further the effect of fibrates on hepatic SR-BI protein
expression by an alternative technique, immunofluorescence microscopy
of SR-BI was performed on livers from control and 0.2% ciprofibrate-treated mice (Fig. 1D). As shown in untreated
control mice, endogenous SR-BI is primarily located in the sinusoidal domain of hepatocytes (Fig. 1D, left panel). No
basal SR-BI signal was detected in the canalicular surface of liver
cells. After receiving ciprofibrate, there was a profound reduction in
hepatic SR-BI protein expression detected by immunofluorescence
analysis (Fig. 1D, middle panel). Note that
immunostaining for SR-BI in liver slices of 0.2% ciprofibrate-treated
mice is almost undistinguishable from those of the SR-BI knockout (Fig.
1D, right panel).
SR-BI Protein Expression in Fibrate-treated Hepatocytes--
To
evaluate whether fibrate treatment alters liver SR-BI protein
expression through a direct action on the hepatocyte, the effect of 100 µM fenofibric acid, the active form of fenofibrate, on
SR-BI protein levels was tested in cultured rat hepatocytes (Fig.
2). Treatment of rat hepatocytes with
fenofibric acid reduced SR-BI protein expression by ~50% compared
with non-treated hepatocytes, indicating that fibrates suppress hepatic
SR-BI protein expression acting in a direct manner on liver parenchymal
cells.

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Fig. 2.
SR-BI protein expression in fibrate-treated
cultured rat hepatocytes. Isolated rat hepatocytes were cultured
for 24 h in the absence (control) or presence of 100 µM fenofibric acid (FF). Hepatocyte
post-nuclear lysates were prepared, subjected to electrophoresis, and
immunoblotted with anti-SR-BI and anti-actin antibodies. Primary
antibody binding was detected by chemiluminescence. SR-BI levels are
expressed as fold change relative to control cells after normalization
for actin levels (*, p < 0.05). The results of this
figure are representative of three independent experiments.
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Hepatic SR-BI mRNA Levels in Mice Treated with
Ciprofibrate--
Fibrate-mediated changes in protein expression in
the liver have been shown so far to be due to changes in target gene
transcriptional activity, secondary to activation of the nuclear
receptor PPAR-
(reviewed in Refs. 19 and 20). Therefore, we tested
whether lowered hepatic SR-BI protein expression induced by fibrates
was also due to decreased gene expression by analyzing SR-BI mRNA levels in livers from control and ciprofibrate-treated animals. Surprisingly, ciprofibrate treatment was not associated with changes in
steady state hepatic SR-BI mRNA levels (Fig.
3), suggesting a post-transcriptional
mechanism for fibrate-dependent regulation of SR-BI protein
expression in the liver.

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Fig. 3.
Hepatic SR-BI mRNA levels in control and
fibrate-treated mice. Wild-type C57BL/6 mice were fed for 7 days
with standard chow diet without (control) and with
ciprofibrate supplementation at increasing doses. Liver samples were
removed, and total RNA was isolated, fractionated by electrophoresis,
transferred to nylon, and hybridized with 32P-labeled SR-BI
and 18 S rRNA cDNA probes, and radioactive bands were measured
with a PhosphorImager. SR-BI mRNA levels are expressed as fold
change relative to chow-fed mice after correction for 18 S rRNA levels
(n = 4). The results of this figure are representative
of two independent RNA expression analyses for each experimental
condition.
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Fibrate Responsiveness of Hepatic SR-BI Expression in
PPAR-
-deficient Mice--
The next important question to address
was whether the down-regulation of hepatic SR-BI expression by fibrates
required the presence of PPAR-
or whether this effect was
PPAR-
-independent. Thus, fibrate regulation of hepatic SR-BI protein
expression was analyzed in mice lacking PPAR-
. Wild-type and
PPAR-
-deficient mice were treated with 0.2% fenofibrate for 14 days, and hepatic SR-BI protein levels were analyzed by immunoblot
(Fig. 4). Fibrate administration
decreased SR-BI protein in livers of wild-type mice by 72%, whereas no
change was observed in PPAR-
-deficient animals. The lack of a
regulatory effect on hepatic protein SR-BI levels in fibrate-treated
PPAR-
-deficient mice shows unequivocally that PPAR-
is both
involved and required for the fibrate responsiveness of SR-BI protein
expression.

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Fig. 4.
Hepatic SR-BI protein expression in
fibrate-treated control and PPAR- -deficient
mice. Control (PPAR- +/+) and PPAR- -deficient (PPAR-
/ ) mice were fed for 14 days with standard chow diet without
(control) or with supplementation with 0.2% fenofibrate
(FF). Liver samples were removed, and total membranes were
prepared, fractionated by electrophoresis, transferred to
nitrocellulose, and immunoblotted with an anti-SR-BI antibody. Primary
antibody binding was detected by an appropriate secondary antibody and
chemiluminescence. Equal protein loading was checked by Ponceau Red
staining of the nitrocellulose membrane before incubation with
antibodies. SR-BI levels are expressed as fold change relative to
chow-fed PPAR- expressing mice (n = 4; *,
p < 0.001).
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Hepatic CLAMP Protein and mRNA Levels in Fibrate-treated
Mice--
CLAMP (for carboxyl-terminal linking
and modulator protein, also known
as PDZk1) is a multiple PDZ domain-containing protein, which has
recently been shown to interact through its most amino-terminal PDZ
domain with the cytoplasmic carboxyl-terminal tail of SR-BI (29). By
coexpressing SR-BI and CLAMP in Chinese hamster ovary cells, steady
state levels of SR-BI protein were significantly higher in the presence
of CLAMP (29), suggesting its role in stabilizing SR-BI protein expression.
Next, we evaluated whether changes in CLAMP expression may provide a
potential mechanism for the fibrate-dependent
post-transcriptional regulation of SR-BI protein expression in murine
liver. As was the case for SR-BI, CLAMP protein levels were drastically
decreased in the liver by fibrate administration (Fig.
5A), following dose and time
pattern of responses (Fig. 5B) similar to those of SR-BI in
fibrate-treated mice (Fig. 1, B and C). To
evaluate further whether decreased hepatic CLAMP protein levels were
due to transcriptional repression of the CLAMP gene by fibrates, steady
state CLAMP mRNA levels in livers of control and fibrate-treated
mice were measured by Northern blot analysis (Fig. 5C).
Fibrates did not produce any alteration in hepatic CLAMP mRNA
levels, indicating that neither SR-BI nor CLAMP were subjected to
transcriptional regulation as a result of PPAR-
activation by
fibrates.

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Fig. 5.
Hepatic CLAMP expression analysis in control
and fibrate-treated mice. Wild-type C57BL/6 mice were fed with
standard chow diet without (control) and with
supplementation with fibrates as follows: A, 0.2%
ciprofibrate (CF) or 0.2% fenofibrate (FF) for 7 days; B, ciprofibrate at increasing doses for 7 days or
0.02% ciprofibrate for increasing times; C, 0.2%
ciprofibrate for 7 days. Liver samples were removed and processed for
immunoblotting and Northern blotting as described under "Experimental
Procedures." A and B, primary antibody binding
was detected by appropriate secondary antibodies for chemiluminescence.
C, hybridization with 32P-labeled probes was
analyzed by a PhosphorImager. CLAMP protein and mRNA levels are
expressed as fold change relative to chow-fed mice after normalization
for -COP and 18 S rRNA signals, respectively (n = 4; *, p < 0.01). The results of this figure are
representative of 2 independent protein and RNA expression analyses for
each experimental condition.
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Plasma Lipoprotein Analysis and Recombinant Adenoviral Transfer of
SR-BI to Fibrate-treated Mice--
Due to its ability to mediate
selective HDL cholesterol uptake, the experimental manipulation of
hepatic SR-BI levels in mice is tightly associated with changes in HDL
metabolism (6-9). In order to assess a potential physiological
relevance of fibrate-mediated hepatic SR-BI repression with regard to
HDL metabolism, we performed FPLC lipoprotein cholesterol analyses in
mice fed fibrate diets. Fig. 6 shows that
ciprofibrate (A) and fenofibrate (B) treatment led to the appearance of a broader HDL cholesterol peak shifted to
earlier FPLC fractions, indicating the presence of more disperse and
larger HDL particles. In fact, the HDL-C peak of ciprofibrate (Fig.
6A, gray circles) and fenofibrate (Fig.
6B, gray circles) groups eluted one fraction
ahead of the HDL peak detected in the control group (Fig. 6, open
circles). Besides an altered HDL size distribution, there was a
substantial reduction in VLDL cholesterol in fibrate-treated animals
reflecting the well known effect of PPAR-
activation on
triglyceride-rich lipoprotein synthesis and catabolism (18).

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Fig. 6.
Plasma lipoprotein cholesterol profiles of
control and fibrate-treated mice after infection with an SR-BI
recombinant adenovirus. Wild-type C57BL/6 mice were fed for 9 days
with a standard chow diet without (control) or with
supplementation with 0.2% ciprofibrate (CF, A)
or 0.2% fenofibrate (FF, B). At day 4, one group
of mice treated with either ciprofibrate or fenofibrate were infected
with murine sr-bi recombinant adenovirus
(Ad.mSR-BI). At day 8, mice were fasted overnight prior to
blood sampling for plasma lipoprotein cholesterol profiling by FPLC and
liver harvesting for SR-BI expression analysis by Western blotting
(insets). Chromatograms are representative of at least two
independent pooled (n = 4) plasma FPLC analyses for
each experimental condition. The approximate elution positions of
VLDL, intermediate density lipoproteins
(IDL)/LDL, and HDL are
indicated.
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By using molecular size monitoring by a non-denaturing electrophoretic
mobility assay of density gradient-purified HDL, similar effects of
fibrates on HDL size were reported in wild-type and human apoA-I
transgenic mice (25). That study also showed that the induction of PLTP
expression is required for the fibrate-mediated HDL particle
enlargement. Because SR-BI is another key regulator of HDL particle
size (6), we tested whether normalizing SR-BI hepatic protein levels by
recombinant adenoviral sr-bi gene transfer might restore HDL
particle size in fibrate-treated mice. FPLC lipoprotein cholesterol
profiling shows that restoring hepatic SR-BI protein expression (Fig.
6, insets) abolished the fibrate-induced HDL enlargement
(Fig. 6, black circles) with a concomitant reduction in HDL
and LDL cholesterol levels. Ciprofibrate-treated mice infected with an
adenovirus carrying the control gene lacZ showed an almost identical FPLC profile to that of mice treated with fibrates only (data
not shown), establishing that the reversal of HDL enlargement was
mediated specifically by SR-BI and not by an artifact due to the
adenoviral infection. These findings suggest that SR-BI down-regulation
plays a role in determining increased HDL particle size following
PPAR-
activation by fibrates.
 |
DISCUSSION |
This study demonstrates that SR-BI and the SR-BI-interacting
protein CLAMP are down-regulated by fibrates in the murine liver by a
novel post-transcriptional PPAR-
-dependent mechanism.
Fibrate-mediated reduced hepatic SR-BI expression correlated with
enlarged HDL particle size, which was reverted by normalizing hepatic
SR-BI levels by recombinant adenoviral gene transfer. Taken together, these findings are consistent with a functionally relevant effect of
fibrate-dependent hepatic SR-BI regulation on HDL
metabolism in mice.
Even though SR-BI is a key regulator of HDL metabolism (reviewed in
Refs. 3-5), the role of PPAR-
activation on controlling SR-BI
expression in the liver, a major site of PPAR-
and SR-BI expression
as well as selective HDL cholesterol uptake, had not been thoroughly
investigated. In clear contrast to the effect observed in macrophages
(28), ciprofibrate and fenofibrate led to a marked reduction of
immunodetectable SR-BI in the liver of treated mice in a dose- and
time-dependent manner (Fig. 1). More intriguingly, hepatic
SR-BI mRNA levels remained unchanged (Fig. 3), suggesting a
post-transcriptional mechanism for SR-BI protein down-regulation by
fibrates. SR-BI protein was also reduced by fibrate treatment in
isolated rat hepatocytes (Fig. 2), clearly showing that the in
vivo data were not an indirect systemic effect of fibrates but
rather involved a defined molecular mechanism of action of these drugs
on the hepatic cells.
Because SR-BI was post-transcriptionally down-regulated, it was
tempting to speculate that PPAR-
would not be required for the
effect of fibrates on hepatic SR-BI expression. However, the post-transcriptional regulation of SR-BI indeed required normal PPAR-
expression as shown by the lack of effect of fibrate treatment on hepatic SR-BI protein levels of PPAR-
- deficient mice (Fig. 4).
Post-transcriptional control of SR-BI expression was originally suggested by the initial characterization of its expression pattern in
different tissues (34) and has later been reported in a variety of
experimental models. In apoE-deficient mice, there was a 2.3-fold induction of hepatic SR-BI protein without changes in SR-BI mRNA (39). Similarly, Witt et al. (40) demonstrated that hepatic SR-BI protein was strongly induced (11-fold) in rats fed a vitamin E-deficient diet and was down-regulated in HepG2 cells incubated with a
vitamin E-enriched culture medium, in both cases without concomitant
changes in SR-BI mRNA. In nephrotic rats, there was also a decrease
in hepatic SR-BI protein, but SR-BI mRNA levels remained unaltered
(41). Finally, intestinal SR-BI protein, but not mRNA, is
down-regulated in several experimental models of impaired enterohepatic
circulation, such as bile-diverted rats, Mdr2-deficient mice, and
cholesterol-7
-hydroxylase-deficient mice (42). Whether altered
PPAR-
signaling might be underlying the post-transcriptional
regulation of SR-BI under some of these experimental conditions remains
to be explored. However, the current work clearly indicates that
hepatic SR-BI regulation is commanded by a
PPAR-
-dependent fibrate-activated event possibly due to transcriptional modulation of a yet unrecognized gene that controls expression/stability of SR-BI protein itself and/or other proteins involved in the SR-BI pathway.
Interestingly, CLAMP (a hepatic protein potentially involved in SR-BI
stabilization in hepatocyte plasma membrane (29)) showed a similar
regulated expression as SR-BI in fibrate-treated animals (Fig. 5). This
is the first example of pharmacological regulation of CLAMP expression
in vivo. By using site-directed mutagenesis of SR-BI within
the CLAMP-interacting domain, Silver (43) has shown recently the
critical requirement of SR-BI/CLAMP interaction for adequate expression
of SR-BI in the cell surface of hepatocytes. On the other hand, SR-BI
expression is not required for hepatic CLAMP expression, because CLAMP
protein levels are normal in livers of SR-BI-deficient mice (data not
shown). We speculate that the simultaneous post-transcriptional
down-regulation of hepatic CLAMP and SR-BI expression by fibrates might
have been caused by modulation of a yet undiscovered PPAR-
target
gene that controls SR-BI and CLAMP protein synthesis, interaction, intracellular trafficking, or degradation as a protein-protein complex,
or by two completely independent regulatory events on these proteins
elicited by fibrates. As reported previously for estrogen and androgen
receptors (44-46), PPAR-
could also be modulating a signal
transduction pathway through a non-genomic activity that results in
decreased SR-BI and CLAMP protein levels.
The effect of fibrates on hepatic SR-BI and CLAMP protein expression is
indeed the first reported case in which fibrate treatment-induced changes in the levels of proteins expressed in the liver are not associated with concomitant variations in their corresponding mRNAs. In this regard, our study also indicates that gene
expression analyses associated with PPAR-
activation might have been
biased by only looking at steady state mRNA levels of target genes
rather than protein levels or functional activities encoded by those genes. In fact, a preliminary study (47) has reported no effect of
treatment with PPAR-
agonists on hepatic SR-BI mRNA levels in
primary rat hepatocytes. Our analysis on SR-BI regulation induced by
fibrates emphasizes the potential relevance of proteomic studies regarding regulation by pharmacological or physiological activators of
PPAR-
in particular as well as other nuclear receptors and transcription factors.
Mice treated with the PPAR-
agonists ciprofibrate or fenofibrate
exhibited enlarged HDL particle sizes, which were normalized when
hepatic SR-BI protein levels were restored by recombinant adenoviral
SR-BI gene transfer in fibrate-treated mice (Fig. 6, A and
B). These findings are consistent with a potential role of
SR-BI in the altered HDL particle size distribution produced by fibrate
treatment besides the up-regulation of PLTP (25). The absolute
requirement of PLTP up-regulation for fibrate-induced HDL particle
enlargement, as reported previously (25), does not discard the notion
that this phenotype might also be due to the down-regulation of SR-BI,
whose deficiency in mice caused the appearance of bigger and more
heterogeneous HDL particles (6) similar to those found in
fibrate-treated mice. We speculate that SR-BI may contribute to the
appearance of abnormal HDL particles in fibrate-treated PLTP-expressing
mice as a downstream element of the PLTP-mediated HDL remodeling.
However, the hepatic SR-BI pathway for selective cholesterol uptake may
be less relevant in PLTP-deficient mice because this receptor has
higher affinity for lipid-rich large HDL (48). In fact, lipid-poor HDL
from PLTP knockout mice are hypercatabolized most likely through an SR-BI-independent pathway (49). Taken together, these findings suggest
that SR-BI and PLTP may act in concert to modulate HDL size
distribution in wild-type mice, whereas SR-BI is less relevant in
controlling HDL particle size in PLTP-deficient animals.
In summary, we have identified a novel PPAR-
-dependent,
post-transcriptional effect of fibrates over SR-BI expression in murine
liver, which may lead to a better understanding of how these drugs
affect lipoprotein metabolism in vivo.
We thank Monty Krieger for providing
antibodies and support, Valeska Volrath and Verónica
Quiñones for helping with Northern blotting, and Frank J. Gonzalez for sharing the PPAR-
knockout mice.
Published, JBC Papers in Press, January 2, 2003, DOI 10.1074/jbc.M211627200
The abbreviations used are:
HDL-C, HDL
cholesterol;
apo, apolipoprotein;
CETP, cholesteryl ester transfer
protein;
CLAMP, carboxyl-terminal linking and modulator protein;
FPLC, fast performance liquid chromatography;
HDL, high density lipoproteins;
LDL, low density lipoproteins;
PPAR-
, peroxisome
proliferator-activated receptor-
;
PLTP, phospholipid transfer
protein;
SR-BI, scavenger receptor class B type I;
VLDL, very low
density lipoproteins;
PBS, phosphate-buffered saline.
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