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Regulation of the proprotein convertase subtilisin/kexin type 9 in intestinal
epithelial cells
Franc¸ois Leblond,
1
Nabil G. Seidah,
2
Louis-Philippe Pre´court,
1
Edgard Delvin,
3
Michel Dominguez,
1
and Emile Levy
1
Departments of
1
Nutrition and
2
Biochemical Neuroendocrinology, Clinical Research Institute of Montre´al;
3
Department
of Biochemistry, Research Center, CHU Sainte Justine, Universite´ de Montre´al, Montre´al, Que´bec, Canada
Submitted 10 July 2008; accepted in final form 26 January 2009
Leblond F, Seidah NG, Pre´court LP, Delvin E, Dominguez
M, Levy E. Regulation of the proprotein convertase subtilisin/
kexin type 9 in intestinal epithelial cells. Am J Physiol Gastrointest
Liver Physiol 296: G805–G815, 2009. First published January 29,
2009; doi:10.1152/ajpgi.90424.2008.—Proprotein convertase sub-
tilisin/kexin type 9 (PCSK9) posttranslationally promotes the deg-
radation of the low-density lipoprotein receptor (LDLr) in hepato-
cytes and increases plasma LDL cholesterol. It is not clear,
however, whether PCSK9 plays a role in the small intestine. Here,
we characterized the patterns of variations of PCSK9 and LDLr in
fully differentiated Caco-2/15 cells as a function of various poten-
tial effectors. Cholesterol (100 M) solubilized in albumin or
micelles significantly downregulated PCSK9 gene (30%, P⬍0.05)
and protein expression (50%, P⬍0.05), surprisingly in concert
with a decrease in LDLr protein levels (45%, P⬍0.05). Cells
treated with 25-hydroxycholesterol (50 M) also displayed signif-
icant reduction in PCSK9 gene (37%, P⬍0.01) and protein (75%
P⬍0.001) expression, whereas LDLr showed a decrease at the
gene (30%, P⬍0.05) and protein (57%, P⬍0.01) levels,
respectively. The amounts of PCSK9 mRNA and protein in Caco-
2/15 cells were associated to the regulation of 3-hydroxy-3-
methylglutaryl-CoA reductase and sterol regulatory element bind-
ing protein-2 (SREBP-2) that can transcriptionally activate PCSK9
via sterol-regulatory elements located in its proximal promoter
region. On the other hand, depletion of cholesterol content by
hydroxypropyl--cyclodextrin upregulated PCSK9 transcripts
(20%, P⬍0.05) and protein mass (540%, P⬍0.001), in parallel
with SREBP-2 protein levels. The addition of bile acids (BA)
taurocholate and deoxycholate to the apical culture medium low-
ered PCSK9 gene expression (25%, P⬍0.01) and raised PCSK9
protein expression (30%, P⬍0.01), respectively, probably via the
modulation of farnesoid X receptor. Furthermore, unconjugated
and conjugated BA exhibited different effects on PCSK9 and
LDLr. Altogether, these data indicate that intestinal PCSK9 is
highly modulated by sterols and emphasize the distinct effects of
BA species.
enterocyte; PCSK9; cholesterol; bile acids; farnesoid X receptor;
SREBP-2
FAMILIAL HYPERCHOLESTEROLEMIA is characterized by elevated
plasma low-density lipoprotein (LDL) and deposition of LDL
in arteries, leading to premature cardiovascular events (15).
This genetically dominant hypercholesterolemia is related to
molecular aberrations in the LDL receptor (LDLr) or its ligand
apolipoprotein B. Recently, mutations in a third gene, propro-
tein convertase subtilisin/kexin type 9 (PCSK9), were associ-
ated to this disease (1). Human PCSK9 gene is ⬃22 kb long,
comprising the promoter region and 12 exons, and it is located
on chromosome 1p32 (49). The gene produces an mRNA of
3,636 bp encoding a 692-aa protein. Although it is proposed
that secreted PCSK9 interacts with LDLr, enters the endocytic
recycling pathway with LDLr, and impairs LDLr pathway by
increasing the degradation of the receptor (5, 37), the precise
molecular basis has yet to be determined.
PCSK9 is mainly expressed in the liver. It is thought to be
involved in liver regeneration, neuronal differentiation/apopto-
sis, and cortical neurogenesis (49), but its primary function
apparently consists in negatively regulating LDLr, likely by a
posttranscriptional manner, e.g., degradation of LDLr in acidic
subcellular compartments, possibly endosomes/lysosomes (23,
30, 49, 50). Even though PCSK9 has been found expressed
substantially in intestine (49), studies have been mostly re-
stricted to the liver (23, 30, 50). However, maintaining cho-
lesterol homeostasis in the body requires accurate metabolic
cross talk between hepatic and intestinal processes to ade-
quately cope with large fluctuations in dietary cholesterol
intake (58), whereas imbalance may lead to elevated LDL
cholesterol levels and increased risks for coronary heart
disease, the main cause of death in Western societies (56).
Notably, the intestine plays a key role in cholesterol balance
in animals and humans (55), constitutes the only site for
absorption of dietary sterols (57), quantitatively represents
the single active location for cholesterogenesis (53, 59), and
remains the second most important organ for the uptake and
degradation of circulating LDL (52). Whether PCSK9 re-
duces the number of LDLr in the enterocyte basolateral
membrane is not known. This information is crucial since
LDLr behavior in absorptive cells does not necessarily
reflect that in hepatocytes.
Poor information is available about the regulation of PCSK9
even though it seems to be an attractive target for lowering
LDL cholesterol (1). In mice, PCSK9 is downregulated by
dietary cholesterol. Conversely, PCSK9 gene expression is
upregulated in mice overexpressing sterol regulatory element
binding proteins (SREBP)-1␣and SREBP-2 (two transcription
factors activated by low levels of intracellular cholesterol)
(24). The nutritional status also modulates PCSK9 expression.
For example, 24-h fasting in mice dramatically decreased
hepatic PCSK9 mRNA and protein levels, which were progres-
sively restored by carbohydrate refeeding (9). Similarly, statins
have been shown to lower LDL by inducing SREBP-2, which
enhances LDLr expression. Moreover, the peroxisome prolif-
Address for reprint requests and other correspondence: E. Levy, GI-Nutri-
tion Unit, CHU Sainte-Justine, 3175 Coˆ te Ste-Catherine, Montreal, Quebec,
Canada, H3T 1C5 (e-mail: emile.levy@recherche-ste-justine.qc.ca).
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.
Am J Physiol Gastrointest Liver Physiol 296: G805–G815, 2009.
First published January 29, 2009; doi:10.1152/ajpgi.90424.2008.
0193-1857/09 $8.00 Copyright ©2009 the American Physiological Societyhttp://www.ajpgi.org G805
erator-activated receptor (PPAR)-␣agonist fenofibrate down-
regulates PCSK9 gene expression and protein synthesis in
controls and not, as expected, in PPAR-␣-deficient mice (31).
Most of the work on PCSK9 regulation has been performed in
relation to the liver, but it must be extended to the small
intestine, an important site for cholesterol homeostasis. There-
fore, the purpose of the present study was to examine the
regulation of PCSK9 in an established Caco-2/15 enterocyte
cell line. An additional aim of the present work was to
determine the expression profile of LDLr and various nuclear
transcription factors.
MATERIALS AND METHODS
Cell culture. Caco-2/15 cells (American Type Culture Collection,
Rockville, MD) were grown at 37°C with 5% CO
2
in minimal
essential medium (MEM) (GIBCO-BRL, Grand Island, NY) contain-
ing 1% penicillin-streptomycin and 1% MEM nonessential amino
acids (GIBCO-BRL) and supplemented with 10% decomplemented
fetal bovine serum (FBS) (Flow, McLean, VA). Caco-2/15 cells
(passages 30 – 40) were maintained in T-75-cm
2
flasks (Corning Glass
Works, Corning, NY). Cultures were split (1:6) when they reached
70 –90% confluence, by use of 0.05% trypsin-0.5 mM EDTA
(GIBCO-BRL). For individual experiments, cells were plated at a
density of 1 ⫻10
6
cells/well on 24.5-mm polycarbonate Transwell
filter inserts with 0.4-m pores (Costar, Cambridge, MA), in MEM
(as described above) supplemented with 5% FBS. The inserts were
placed into six-well culture plates, permitting separate access to the
upper and lower compartments of the monolayers. Cells were cultured
for various periods, including 21 days, at which the Caco-2/15 cells
are highly differentiated and appropriate for lipid synthesis (16, 34,
48). The medium was refreshed every second day.
Cell treatments. Cholesterol and 25-hydroxycholesterol (Sigma-
Aldrich Canada, Oakville, ON) were dissolved in chloroform and
dried under nitrogen. Acetone (0.5 ml) was then added to dissolve the
dried sterol. This solution was slowly added with continuous stirring
to 3 ml of an albumin solution (250 mg of fatty acid-poor BSA in 3
ml of 10% BSA pH 7.4). This mixture was placed under a stream of
nitrogen until the odor of acetone was no longer detectable. Micellar
cholesterol was prepared as described previously (45). The mixed bile
salt micelle, as a vehicle of cholesterol and 25-hydroxycholesterol,
contained 4.8 mM sodium taurocholate and 0.3 mM monoolein. The
micellar solution was warmed to 37°C and stirred vigorously before
use. Albumin- or micelle-bound sterols were mixed to MEM without
FBS (18 h) before being added to the apical compartment. Hy-
droxypropyl--cyclodextrin (Sigma-Aldrich Canada) was directly
dissolved in MEM at a 25 mM concentration and then added to the
apical compartment. Cholate (4.8 mM), chenodeoxycholate (250 M)
and conjugated forms (Calbiochem, Gibbstown, NJ), as well as
deoxycholate (250 M) (ICN Biomedicals, Aurora, OH) and conju-
gated forms (Calbiochem, Gibbstown, NJ) were dissolved in 95%
ethanol and evaporated under nitrogen before being mixed with MEM
at their final concentrations.
Antibodies against PCSK9. The specificity of the Abs (particularly
PCSK9) (43, 46) was evaluated by various methods, including West-
ern blotting following the incubation of the Abs in the presence or
Fig. 1. Influence of cholesterol (solubilized in
albumin) incubated for 24 h on gene and
protein expressions of proprotein convertase
subtilisin/kexin type 9 (PCSK9) and low-den-
sity lipoprotein receptor (LDLr) in intestinal
epithelial cells. Caco-2/15 cells were incu-
bated with fresh medium, allowed to grow and
differentiate, and tested for the effects of cho-
lesterol on transcript levels and protein mass
of PCSK9 and LDLr by RT-PCR and Western
blotting, respectively. The control cells were
incubated with albumin only. Values are ex-
pressed as means ⫾SE for 3 different exper-
iments. *P⬍0.05; **P⬍0.01; ***P⬍
0.001 vs. controls.
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absence of specific antigens, the omission of the primary PCSK9 Ab
in Western blot, and the identification of the human PCSK9 sequences
following immunoprecipitation and SDS-PAGE.
Western blots. To assess the presence and regulation of PCSK9,
LDLR, and SREBP-2, Caco-2/15 cells were homogenized and ade-
quately prepared for Western blotting as described previously (33).
The Bradford assay (Bio-Rad) was used to estimate protein concen-
tration. Proteins were denatured in sample buffer containing SDS and
DTT (Thermo Scientific, Rockford, IL), separated on a 7.5% SDS-
PAGE and electroblotted onto Hybond-C extra nitrocellulose mem-
branes (Amersham Biosciences, Piscataway, NJ). Nonspecific binding
sites of the membranes were blocked with defatted milk proteins
followed by the addition of primary antibodies directed against the
targeted proteins: -actin (Sigma-Aldrich Canada), LDLr (Research
Diagnostic, Flanders, NJ), PCSK9 (amino acids 31-454) (kind gift of
G. Dubuc and J. Davignon, Clinical Research Institute of Montreal,
University of Montreal, Montreal, QC, Canada), and SREBP-2 (Cayman,
Ann Arbor, MI). The relative amount of primary antibody was
detected with species-specific horseradish peroxidase-conjugated
secondary antibody. Even if identical protein amounts of tissue
homogenates were applied, the -actin protein was used as a
reference protein. Molecular size markers were simultaneously
loaded on gels (data not shown on the figures). Blots were
developed and the mass of proteins were quantified by using an HP
Scanjet scanner equipped with a transparency adapter and soft-
ware. Importantly, the utilization of differential loading and quan-
tification suggested a dynamic range of densitometric measure-
ments.
RT-PCR. Experiments for mRNA quantification as well as for
GAPDH (as a control gene) were performed by using the Eppendorf
Mastercycler Gradient PCR machine (Eppendorf, Westbury, NY) as
reported previously (40, 48). Approximately 30 – 40 cycles of amplifica-
tion were used at 95°C for 30 s, 58°C for 30 s, and 72°C for 30 s. The
following primer sequences were used: farnesoid X receptor (FXR)
forward (5⬘-CATGCGAAGAAAGTGTCAAGAGTGTCG-3⬘) and re-
verse (5⬘-CTTTGTTGTCGAGGTCACTTGTCGCA-3⬘); GAPDH for-
ward (5⬘-GTCCACTGGCGTGTTCACCA-3⬘) and reverse (5⬘-
GTGGCAGTGATGGCATGGAC-3⬘); 3-hydroxy-3-methylglutaryl
(HMG)-CoA reductase forward (5⬘-ACCCTTAGTGGCTGAAACA-
GATACCC-3⬘) and reverse (5⬘-AACTGTCGGCGAATAGATACAC-
CACG-3⬘); LDLr forward (5⬘-TGAGAGGACCACCCTGAGCAAT-3⬘)
and reverse (5⬘-TTACGGCTGTGGAGCTGACCTTTA-3⬘); and
PCSK9 forward (5⬘-AGGACTGTATGGTCAGCACACT-3⬘) and re-
verse (5⬘-CGGGATTCCATGCTCCTTGACTTT-3⬘). Amplicons were
visualized on standard ethidium bromide-stained agarose gels. Under
these experimental conditions relative to RT-PCR, 34 –36 cycles corre-
sponded to the linear portion of the exponential phase. Pilot and previous
studies in our laboratories showed that the RT-PCR, under our experi-
mental conditions, is quantitative employing different amounts of RNA.
Fold induction was calculated by using GAPDH (glyceraldehyde 3-phos-
phate dehydrogenase) as the reference gene.
Fig. 2. Effect of cholesterol (solubilized in bile
acid micelles) incubated for 24 h on gene and
protein expressions of PCSK9 and LDLr intes-
tinal epithelial cells. Caco-2/15 cells were incu-
bated with fresh medium, allowed to grow and
differentiate, and tested for the effects of micel-
lar cholesterol on transcript levels and protein
mass of PCSK9 and LDLr by RT-PCR and
Western blotting, respectively. The control cells
were incubated with bile acid micelles only.
Values are expressed as means ⫾SE for 3
different experiments. *P⬍0.05 vs. micelles.
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Statistical analysis. All values were expressed as means ⫾SE. The
data were evaluated by ANOVA, where appropriate, and the differ-
ences between the means were assessed by the two-tailed Student’s
t-test.
RESULTS
The first issue addressed by our studies was to explore
whether a difference could be noted in the expression of
PCSK9 under the influence of sterols. The incubation of fully
differentiated Caco-2/15 cells with various concentrations of
cholesterol solubilized in albumin resulted in a decrease in the
gene and protein expression of PCSK9. At 100 M cholesterol,
decreases of 25% (P⬍0.001) and 55% (P⬍0.05) were
significantly recorded in mRNA and protein mass levels, re-
spectively (Fig. 1A). When the regulation of LDLr was inves-
tigated, a lower protein expression (⬃60%, P⬍0.001) along
with significantly unchanged mRNA levels was noted (Fig.
1B). Since bile acid (BA)-facilitated cholesterol absorption
occurs in the small intestine in physiological conditions, solu-
tions containing either micelles alone or micelles together with
cholesterol were added to the apical media. Again, a micellar
cholesterol-mediated drop (30 –50%) was recorded in the gene
and protein expression of PCSK9 (Fig. 2A), accompanied with
a similar reduction in LDLr protein expression (Fig. 2B).
Finally, the substitution of cholesterol by 25-hydroxycholes-
terol produced a similar results on PCSK9 (Fig. 3A) and on
both mRNA and protein levels of LDLr (Fig. 3B).
In view of these findings, we assessed protein expression
of nuclear SREBP-2 with an antibody that distinguished the
truncated active nuclear form from its membrane-linked
precursor. Also, since LDLr and HMG-CoA reductase, the
limiting enzyme in cholesterol synthesis, are sensitive to
SREBP-2 and sterol in the small intestine, we determined
HMG-CoA reductase transcription. As illustrated in Fig. 4A,
the administration of micellar cholesterol or 25-hydroxy-
cholesterol to Caco-2/15 cells lowered HMG-CoA reductase
transcripts (30 and 55%, respectively). Moreover, measure-
ment of SREBP-2 revealed a lessening in its protein expres-
sion (Fig. 4B).
We next evaluated how depletion of cholesterol cell content
modulates PCSK9 and LDLr. The extraction of cholesterol was
achieved with hydroxypropyl--cyclodextrin that has been
shown to selectively eliminate cholesterol from the plasma
membrane, in preference to other lipids (28). As illustrated in
Fig. 5, Aand B, hydroxypropyl--cyclodextrin markedly in-
creased the gene and protein expression of PCSK9 and LDLr.
The expression of HMG-CoA reductase and SREBP-2 was
Fig. 3. Effect of 25-hydroxycholesterol (sol-
ubilized in albumin) incubated for 24 h on
gene and protein expressions of PCSK9 and
LDLr in intestinal epithelial cells. Caco-2/15
cells were incubated with fresh medium, al-
lowed to grow and differentiate, and tested for
the effects of 25-hydroxycholesterol on tran-
script levels and protein mass of PCSK9 and
LDLr by RT-PCR and Western blotting, re-
spectively. The control cells were incubated
with albumin only. Values are expressed as
means ⫾SE for 3 different experiments. *P⬍
0.05; **P⬍0.01; ***P⬍0.001 vs. controls.
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upregulated as well (Fig. 5C). To determine whether this
impact is mainly due to cholesterol extraction, Caco-2/15 cells
were supplemented with a combination of hydroxypropyl--
cyclodextrin and micellar cholesterol. The mixture largely
restored the control levels.
Since BA play a significant role in cholesterol metabolism
and are increasingly being appreciated as metabolic integrators
and signaling factors, we subsequently tested their specific
effects on PCSK9. Taurocholate (4.8 mM) reduced mRNA
level of PCSK9 by 25% without affecting LDLr expression
(Fig. 6). On the other hand, the addition of deoxycholate (250 M)
enhanced the protein expression of PCSK9 by 30% (Fig. 7A). LDLr
was more responsive to deoxycholate by showing a ⬃70% and
twofold increase in gene and protein expression, respectively
(Fig. 7B). As noted in Fig. 8, taurocholate slightly diminished
the transcripts of HMG-CoA reductase without affecting
SREBP-2 and FXR gene expression. On the other hand, de-
oxycholate decreased FXR gene expression, enhanced the
protein expression of SREBP-2, and remained without effect
on HMG-CoA reductase (Fig. 8).
Given the different properties of unconjugated and conju-
gated BA, we have examined their effects on the expression of
PCSK9 and LDLr. As noted in Table 1, cholate was more
powerful than taurocholate in reducing PCSK9 mRNA,
whereas glycocholate significantly enhanced it. No significant
modifications were observed in PCSK9 protein mass and LDLr
gene expression following the incubation of Caco-2/15 cells
with cholate, taurocholate, and glycocholate. Cholate was also
able to downregulate LDLr protein mass. Although some
alterations were noted in FXR and SREBP2, they never
reached statistical significance. We also assessed the influence
of chenodeoxycholate, taurochenodeoxycholate, and glycoche-
nodeoxycholate on the expression of PCSK9, LDLr, FXR, and
SREBP2 and could not record significant alterations, except for
glycochenodeoxycholate, that increased LDLr and FXR
mRNA. Finally, we evaluated the impact of the two conjugated
forms of deoxycholate. Taurodeoxycholate and glycodeoxy-
cholate were both capable of reducing PCSK9 mRNA levels.
However, only taurodeoxycholate was able to downregulate
LDLr gene expression. In fact, deoxycholate was more effec-
tive in modulating gene and protein expressions of PCSK9,
LDLr, and SREBP-2.
DISCUSSION
Despite valuable advances, additional studies are clearly
warranted to understand the complex molecular mechanisms
that orchestrate cholesterol homeostasis in the small intes-
tine. A tremendous opportunity is offered by the recent
discovery of PCSK9, which displays various functions,
Fig. 4. Impact of micellar cholesterol and 25-
hydroxycholesterol incubated for 24 h on 3-hy-
droxy-3-methylglutaryl (HMG)-CoA reductase
and SREBP-2 expression in intestinal epithelial
cells. Caco-2/15 cells were incubated with fresh
medium, allowed to grow and differentiate, and
tested for the effects of cholesterol and 25-hy-
droxycholesterol on transcript levels of HMG-
CoA reductase by RT-PCR and sterol regulatory
element binding protein-2 (SREBP-2) by Western
blot. The control cells were incubated with bile
acid micelles. Values are expressed as means ⫾
SE for 3 different experiments. *P⬍0.05; **P⬍
0.01; ***P⬍0.001 vs. micelles.
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including hepatic LDLr degradation. In view of the small
intestine’s high capacity to absorb lipids and elaborate most
of the major lipoprotein classes and considering the well-
known actions of a number of nutrients and hormones on
lipid metabolism and transport at the intestinal level, the
lack of knowledge about the modulation of PCSK9 in the
gut is puzzling. For the first time, the present work at-
tempted to detail the modulation of PCSK9 in intestinal
cells. According to our data, there is a discrete regulation of
PCSK9 from stimuli, originating from apical media, such as
sterols, BA, and cyclodextrin. Apparently, PCSK9 and
LDLr are stimulated or downregulated in a similar fashion
via SREBP-2 transcription factor. Furthermore, in our ex-
periments, we have supplemented Caco-2/15 cells with
sterols or caused the depletion of cholesterol cell content
using hydroxypropyl--cyclodextrin, an approach that is
both rapid and highly efficient (18, 19). In addition, hy-
droxypropyl--cyclodextrin is entirely surface acting, so,
apart from removing cholesterol, it has a minimal effect on
cell membranes (25). Our findings clearly showed that
provision of sterols to enterocytes downregulated the gene
and protein expression of PCSK9. Conversely, the lessening
Fig. 5. Regulation of PCSK9, LDLr, HMG-
CoA reductase, and SREBP-2 expression by
hydroxypropyl--cyclodextrin in intestinal
epithelial cells. Caco-2/15 cells were incu-
bated with fresh medium, allowed to grow and
differentiate, and tested for the effects of hy-
droxypropyl--cyclodextrin on transcript lev-
els and protein mass of PCSK9 and LDLr by
RT-PCR and Western blotting, respectively.
The transcripts of HMG-CoA reductase and
protein mass of SREBP-2 were also assessed.
Values are expressed as means ⫾SE for 3
different experiments. *P⬍0.05; **P⬍
0.01; ***P⬍0.001 vs. controls. †⬍0.05;
‡⬍0.01 vs. cyclodextrin.
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of cholesterol cell content enhanced PCSK9 transcription. In
similar fashion, PCSK9 expression was highly downregu-
lated by dietary cholesterol in mouse liver (38). This is also
consistent with the demonstration that PCSK9 expression
was strongly induced by statins in a dose-dependent manner
in HepG2 cells and in human primary hepatocytes, likely a
result of the cholesterol-lowering effect of the drug, and that
this induction was efficiently reversed by mevalonate (11).
Altogether, these studies pointed out that 1) the regulation
of PCSK9 expression is dependent on the presence or
absence of sterols not only in hepatocytes, but also in
intestinal epithelial cells, and 2) the modulation of PCSK9
by sterols, which are supplied either exogenously or endo-
genously, is achieved at the transcription level.
It has been reported that gain-of-function mutations in
PCSK9 have a 23% decreased level of cell surface expression
of LDL receptors and a 38% decrease in internalization of
LDL, whereas loss-of-function mutations are associated with a
16% increased level of cell surface LDLr and a 35% increased
internalization of LDL (7). Apparently, the catalytic activity of
PCSK9 appears not to be required for LDLr degradation, but it
is essential for activation and secretion of PCSK9 (10, 22, 39).
In our work, the protein expression of LDLr showed similar
changes to PCSK9 in Caco-2/15 cells with sterol supplemen-
tation or under the sterol-depleted conditions. Considering that
the function of PCSK9 is to promote the degradation of LDLr
(23), as noted before (3), the parallel change in LDLr to
PCSK9 by sterols is surprising. However, previous studies
have reported a similar behavior of the two proteins in HepG2
cells (26). One might consider that these cells lack a compo-
nent or machinery necessary for the coordination of the ex-
pression of these proteins, but other research groups have not
found a reciprocal regulation of PCSK9 and LDLr. For exam-
ple, inhibition of squalene synthase upregulates PCSK9 and
LDLr expression in rat liver (4) and there was no reverse
relation of PCSK9 and LDLr in rat liver cell line (17). PCSK9
was identified as one of the genes that are regulated by
SREBPs (24, 38). The SREBPs are members of the basic
helix-loop-helix leucine zipper family of transcription factors
that regulate the expression of the target genes by binding to
the sterol-regulatory elements (SRE) in their promoter regions
(6). Dubuc et al. (11) reported that the transcription of PCSK9
was increased by statins, most likely through SREBP-2 acti-
vation in primary human hepatocytes, and proposed the impor-
tance of conserved SRE in the promoter region of mouse, rat,
and human PCSK9 genes. Thereafter, the expression of PCSK9
was found dependent on the absence or presence of sterols via
SRE in the minimal promoter region of the human PCSK9 gene
by both SREBP-1 and SREBP-2 in HepG2 cells, and it was
proposed that the predominant transcriptional regulator of
PCSK9 by cholesterol is SREBP-2 in vivo (26). Accordingly,
our data on intestinal epithelial cells showed that the adminis-
tration of sterols reduced SREBP-2 transcripts while decreas-
ing PCSK9 gene and protein expression. On the other hand,
depletion of cholesterol content in Caco-2/15 cells by cyclo-
dextrin raised the levels of mRNA and protein mass of PCSK9
Fig. 6. Modulation of PCSK9 and LDLr gene
and protein expression by 4.8 mM taurocholate
incubated for 24 h in intestinal epithelial cells.
Caco-2/15 cells were incubated with fresh me-
dium, allowed to grow and differentiate, and
tested for the effects of taurocholate on tran-
script levels and protein mass of PCSK9 and
LDLr by RT-PCR and Western blotting, re-
spectively. Values are expressed as means ⫾
SE for 3 different experiments. *P⬍0.01 vs.
controls.
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and LDLr. As expected, the changes of SREBP-2 and PCSK9
in Caco-2/15 cells paralleled the alterations in mRNA encoding
HMG-CoA reductase, the key cholesterol biosynthetic enzyme.
These results might suggest that PCSK9 would be under the
control of SREBP-2 in Caco-2/15 cells, as is the case for LDLr
and HMG CoA reductase (12–14).
BA are amphiphilic molecules synthesized from cholesterol
exclusively in the liver, represent the major route for removal
of excess cholesterol from the body, and are essential for
effective transport of dietary fat (29). In addition to their
traditional role in dietary lipid absorption and cholesterol
homeostasis, it has become clear now that BA constitute
versatile signaling molecules endowed with systemic endo-
crine functions. In fact, BA are ligands for G protein-coupled
receptors and modulate several nuclear hormone receptors (27,
35, 36). Through activation of these diverse signaling path-
ways, BA have been shown to regulate not only their own
synthesis and enterohepatic recirculation, but also triglyceride,
cholesterol, and glucose homeostasis (51). Given the broad
relationship of BA with multiple pathways and their recent link
with PCSK9 in hepatocytes (32, 44), we decided to investigate
their impact on PCSK9 in the intestine. In the present experi-
ments, we employed taurocholate and deoxycholate, two pow-
erful BA. According to our data in Caco-2/15 cells, tauro-
cholate brought about a decrease in the gene expression of
PCSK9, whereas deoxycholate enhanced that of PCSK9 and
LDLr along with a lessening of FXR mRNA. The distinct
effects of BA on PCSK9 gene expression were also noted in
another study on human hepatocytes that showed a repression
by chenodeoxycholic acid, an induction by deoxycholic acid,
and no influence with cholic acid and ursodeoxycholic acid
(32). Further work is needed to explore whether these separate
impacts on PCSK9 may be due to the potential activation by
BA of other nuclear receptors such as pregnane X receptor,
constitutive and rostane receptor, and vitamin D receptor in
addition to FXR (54, 60).
Chenodeoxycholate and cholate are the two primary BA in
humans and are conjugated mainly to glycine and taurine (21).
Given their hydrophilic-hydrophobic properties, unconjugated
and conjugated BA may differ in their various actions, includ-
ing membrane permeability, lipid solubilization, intracellular
signaling, and DNA synthesis (2, 8, 20, 35, 41, 47). We have
therefore assessed the modulation of unconjugated and conju-
gated BA to taurine or glycine. Our findings indicate the high
ability of cholate, taurodeoxycholate, and glycodeoxycholate
to alter the gene expression of PCSK9. Cholate and taurode-
oxycholate also diminished LDLr protein content and mRNA,
respectively, in Caco-2/15 cells. The effects of cholate were
superior to those of taurocholate and quite divergent from those
of glycocholate. On the other hand, chenodeoxycholate and tau-
rochenodeoxycholate were ineffective in modulating PCSK9 and
LDLr. Only the glycochenodeoxycholate displays a capacity to
Fig. 7. Modulation of PCSK9 and LDLr
gene and protein expression by 250 M de-
oxycholate incubated for 24 h in intestinal
epithelial cells. Caco-2/15 cells were incu-
bated with fresh medium, allowed to grow
and differentiate, and tested for the effects of
deoxycholate on transcript levels and protein
mass of PCSK9 and LDLr by RT-PCR and
Western blotting, respectively. Values are ex-
pressed as means ⫾SE for 3 different exper-
iments. *P⬍0.05; **P⬍0.01; ***P⬍
0.001 vs. controls.
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upregulate LDLr and FXR mRNA. However, we were not able to
show LDLr mRNA upregulation by chenodeoxycholate, previ-
ously reported by Nakahara et al. (42), presumably because of the
difference in experimental techniques. Additional studies are nec-
essary to uncover novel mechanisms by which specific BA mod-
ulate PCSK9 and LDLr. This in turn may have far reaching
consequences in the control of the overall body metabolism
homeostasis and the multiorgan implications.
ACKNOWLEDGMENTS
The authors thank Schohraya Spahis, Carole Garofalo, E
´milie Grenier, and
Genevie`ve Lalonde for helpful technical assistance.
GRANTS
This study was supported by the Canadian Institutes of Health Research
(Grant MOP 49433 to E. Levy and 36496 to N. G. Seidah).
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Table 1. Influence of unconjugated and conjugated bile acids on PCSK9, LDLr, FXR, and SREBP2
Bile Acids
PCSK9 Expression LDLr Expression FXR SREBP-2
Gene Protein Gene Protein Gene Protein
Cholate 0.17⫾0.04‡1.06⫾0.05 0.55⫾0.29 0.13⫾0.05‡1.75⫾0.28 0.17⫾0.05
Taurocholate 0.72⫾0.04 0.99⫾0.05 1.03⫾0.10 0.88⫾0.07 0.96⫾0.25 1.37⫾0.47
Glycocholate 1.45⫾0.07†1.23⫾0.09 1.27⫾0.07 1.05⫾0.13 1.93⫾0.13 1.00⫾0.04
Chenodeoxycholate 0.99⫾0.01 1.07⫾0.05 0.98⫾0.02 0.76⫾0.09 1.23⫾0.13 0.89⫾0.03
Taurochenodeoxycholate 1.06⫾0.03 1.11⫾0.13 1.12⫾0.05 0.93⫾0.16 1.54⫾0.12 0.78⫾0.08
Glycochenodeoxycholate 1.26⫾0.11 0.92⫾0.04 1.32⫾0.11†0.71⫾0.06 2,18⫾0.18†0.76⫾0.00
Deoxycholate 1.13⫾0.09 1.51⫾0.11* 1.83⫾0.22* 1.98⫾0.15†0.66⫾0.16 1.67⫾0.20
Taurodeoxycholate 0.37⫾0.16‡0.99⫾0.26 0.58⫾0.14* 0.59⫾0.26 0.72⫾0.33 0.87⫾0.17
Glycodeoxycholate 0.60⫾0.05†1.20⫾0.06 1.01⫾0.16 1.10⫾0.24 1.12⫾0.18 1.07⫾0.24
Data are expressed as the ratio between the gene of interest 关proprotein convertase subtilisin/kexin type 9 (PCSK9), low-density lipoprotein receptor (LDLr),
farnesoid X receptor (FXR), sterol regulatory element binding protein-2 (SREBP2)兴and the internal reference gene 关GAPDH (for mRNA) and -actin (for protein
mass)兴. Values were compared to respective controls equivalent to 1 and are expressed as means ⫾SE for 3 different experiments. *P⬍0.05; †P⬍0.01;
‡P⬍0.001 vs. control.
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