Download Increased Secretion of Lipoproteins in Transgenic Mice Expressing

Increased Secretion of Lipoproteins in Transgenic Mice
Expressing Human D374Y PCSK9 Under Physiological
Genetic Control
Bronwen Herbert, Dilipkumar Patel, Simon N. Waddington, Emily R. Eden, Alexandra McAleenan,
Xi-Ming Sun, Anne K. Soutar
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Objective—To produce transgenic mice expressing the D374Y variant of the human proprotein convertase subtilisin/kexin
type 9 (PCSK9) gene at physiological levels to investigate the mechanisms causing hypercholesterolemia and
accelerated atherosclerosis.
Methods and Results—A bacterial artificial chromosome containing PCSK9 and its flanking regions was modified to
introduce the D374Y mutation and a C-terminal myc2 tag. Transgenic mice that expressed 1 copy of the mutant or
wild-type (WT) PCSK9 bacterial artificial chromosome were produced. Human PCSK9 mRNA was expressed at levels
comparable to endogenous pcsk9 and with the same tissue specificity. The expression of D374Y or WT human PCSK9
increased the serum cholesterol level and reduced hepatic low-density lipoprotein receptor protein levels in the
transgenic mice compared with bacterial artificial chromosome–negative controls; however, the effects were more
marked in D374Y mice. The effect of a high-cholesterol diet on increasing serum cholesterol level was greater in D374Y
mice, and atherosclerotic plaques after 15 weeks were more extensive in mice expressing D374Y than in WT PCSK9.
D374Y mice secreted more triglyceride-rich lipoproteins into the circulation than WT mice.
Conclusion—The expression of human D374Y PCSK9 at physiological levels produced a phenotype that closely matched
that found in heterozygous D374Y patients and suggested that reduced low-density lipoprotein receptor activity is not
the sole cause of their hypercholesterolemia. (Arterioscler Thromb Vasc Biol. 2010;30:1333-1339.)
Key Words: atherosclerosis 䡲 genetically altered mice 䡲 hyperlipoproteinemia 䡲 lipoproteins 䡲 receptors
I
n 2003, linkage analysis in 2 families with autosomal
dominant familial hypercholesterolemia (FH) of unknown
etiology showed that variation in a novel gene, proprotein
convertase subtilisin/kexin type 9 (PCSK9), cosegregated
with hypercholesterolemia.1 The gene, which encodes a
putative proprotein convertase, was first identified as a gene
that was upregulated in neural cells undergoing apoptosis
(neural apoptosis–regulated convertase-1)2; thus, no underlying causative mechanism for hypercholesterolemia was apparent. Measurement of lipoprotein turnover in 2 patients
heterozygous for a PCSK9 mutation suggested that apolipoprotein B (apoB) synthesis was increased.3 Also, microarray analyses of global gene expression in mice fed a highcholesterol diet4 or overexpressing the transcription factor
SREBP5 revealed that expression of this gene was regulated
by sterols and that it was, therefore, likely to play some role
in cholesterol homeostasis. Adenoviral-mediated expression
of PCSK9 in the liver of mice resulted in reduction of hepatic
low-density lipoprotein (LDL) receptor protein, with no
change in the LDL receptor (LDLR) mRNA6; however, there
was no apparent difference between mice expressing wildtype (WT) and mutant PCSK9.7 On the other hand, genetic
ablation of pcsk9 in mice resulted in increased LDLR levels
and a reduced plasma cholesterol level8; in the general
population, heterozygosity for a null mutation in PCSK9 is
associated with reductions in serum cholesterol level and the
risk of coronary heart disease.9
See accompanying article on page 1279
Thus, it rapidly became clear that PCSK9 normally regulates LDLR levels and thereby influences serum cholesterol
level, and that the rare mutations found in patients with FH
are gain of function. These observations implied that PCSK9
was a target for lipid-lowering therapy. With the added
incentive that inhibition of PCSK9 would act in concert with
statins rather than replace them, research was conducted to
understand the basis of action of PCSK9.10 Briefly, it has
emerged that PCSK9 is synthesized in the liver, undergoes
autocleavage to produce a prosegment that remains associ-
Received on: January 27, 2010; final version accepted on: April 23, 2010.
From the Medical Research Council Clinical Sciences Centre (B.H., D.P., E.R.E., A.M., X.-M.S., and A.K.S.), Imperial College London, London,
England; and the Institute for Women’s Health (S.N.W.), University College London. Dr Eden is now with the UCL Institute of Ophthalmology,
University College London.
Correspondence to Anne K. Soutar, PhD, Medical Research Council Clinical Sciences Centre, Imperial College London, Hammersmith Hospital,
Ducane Road, London W12 0NN, England. E-mail [email protected]
© 2010 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org
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DOI: 10.1161/ATVBAHA.110.204040
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Arterioscler Thromb Vasc Biol
July 2010
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ated with the protein, and is then secreted. Circulating PCSK9
binds to the LDLR on the cell surface and is cointernalized
through clathrin-coated pits,11 although other autosomal recessive hypercholesterolemia (ARH)-independent mechanisms may also exist.12 At the acid pH of endosomes, PCSK9
binds more tightly and prevents dissociation,13 with the result
that the LDLR cannot recycle and is degraded. Gain-offunction PCSK9 mutants bind with even higher affinity than
WT PCSK9.13
We showed that the D374Y mutation in PCSK9 was
associated with a particularly severe clinical phenotype.14
This was characterized in 4 apparently unrelated families of
UK origin by an earlier onset of both severe hypercholesterolemia and coronary heart disease and a poorer response to
lipid-lowering therapy with statins compared with patients
with FH and null mutations in LDLR15; this was subsequently
confirmed elsewhere16. Surprisingly, LDL from these patients
differed in size and composition not only from normal LDL
but also from LDL from LDLR patients with FH; the LDL
was smaller, and there was a lower cholesterol to protein
ratio. Furthermore, PCSK9 LDL competed less well for
binding to the LDLR on cultured skin fibroblasts.15 We also
showed that cultured rat hepatocytes (McArdle RH7777)
expressing human D374Y PCSK9 secreted more apoB100
than untransfected cells or cells expressing WT PCSK9.14
Together, these data suggested that gain-of-function PCSK9
mutations might, in addition to reducing LDLR protein, also
increase apoB secretion, thereby further contributing to hypercholesterolemia. Others have reported that they failed to
observe increased apoB synthesis in mice overexpressing
normal PCSK9 from adenoviral vectors or cDNA transgenes
driven by powerful promoters, although 1 study17 showed that
mice that fasted overexpressed PCSK9 and had an increased
very-low-density-lipoprotein output. However, to our knowledge, no studies have compared WT with D374Y PCSK9
expressed at more physiological levels. Herein, we produced
transgenic mice expressing human WT or D374Y PCSK9
from a single integrated copy of a bacterial artificial chromosome (BAC) containing all intergenic regions between
PCSK9 and adjacent flanking genes; therefore, the exogenous
PCSK9 is expressed under its own genetic control. We show
that mice expressing D374Y PCSK9 secrete more lipoproteins than mice expressing WT PCSK9 or ldlr⫺/⫺ mice and
that atherosclerotic plaques are induced by cholesterol feeding of mice expressing D374Y but not WT PCSK9.
Methods
An expanded “Methods” section can be found in the supplemental
materials (available online at http://atvb.ahajournals.org).
Generation of PCSK9 Transgenic Mice
Human WT and D374Y myc2-tagged PCSK9 cDNA was introduced
by homologous recombination18 into the exon1-intron1 region of
PCSK9 in a BAC containing human PCSK9. Transgenic founders
and offspring were identified by amplification of PCSK9 cDNA.
Transgenic mice were backcrossed onto the C57BL6J background
for at least 5 generations, and BAC-negative littermates served as
controls. Unless otherwise stated, all experiments were performed on
male mice at the ages indicated in the legends to the figures.
Measurement of Lipoprotein Secretion
Mice fasted overnight, were anesthetized with isoflurane, and then
injected intravenously with 10% (vol/vol) tyloxapol in saline (500␮g/g body weight).17 Blood samples were collected from the tail
vein at intervals after injection.
Statistics
Data are expressed as mean⫾SD. Differences between multiple
groups were compared by 1-way ANOVA, followed by pairwise
comparisons using the Bonferroni correction. Direct pairwise comparisons with independent controls were analyzed by the 2-tailed
unpaired t test (Prism and GraphPad Software).
Results
Production of Transgenic Mice Expressing
Single-Copy WT and D374Y PCSK9
The first aim of this study was to produce mice expressing
WT or D374Y mutant PCSK9 under physiological control
and at a low copy number as a model of patients heterozygous
for PCSK9 gain-of-function mutations. A BAC containing
the entire human PCSK9 gene as the only entire coding gene
was modified by recombination to express either WT or
D374Y human PCSK9 with a carboxy-terminal myc2 tag and
under its own genetic control (supplemental Figure I). Random integration of the BAC constructs into the genome
produced 3 transgenic mouse lines, 1 expressing WT PCSK9
and 2 expressing D374Y PCSK9. All the transgenic mice
showed normal fertility, and there was no significant difference in their size or weight gain compared with BACnegative littermates.
Transgenic mice were identified by analysis of genomic
DNA by PCR (supplemental Figure IIA); the mutant D374Y
transgene was distinguished from WT by digestion with AluI
of a 177-bp PCR product across the site of the mutation.14
Phosphorimager quantification of Southern blots probed with
cDNA to human PCSK9 and the single copy gene daf1
(supplemental Figure IIB) suggested that there was a single
copy of the WT human PCSK9 in the genome of the
WT-PCSK9 transgenic mice, 1 copy of the D374Y PCSK9 in
1 of the D374Y mouse lines (D374Y low), and 5 copies in the
other D374Y mouse line (D374Y high).
Expression of WT and D374Y PCSK9 in the Liver
and Small Intestine
To determine the expression of the transgenes and their
possible effect on endogenous pcsk9 expression, we measured endogenous pcsk9 and transgenic PCSK9 mRNA in the
livers of the 3 transgenic mouse lines and in their BACnegative littermate controls by quantitative RT-PCR (supplemental Table I). There was no significant difference
(P⬎0.05) in endogenous pcsk9 mRNA between the 4 groups
(Figure 1), indicating that expression of the transgene at
physiological levels had no effect on endogenous pcsk9
expression. The amount of PCSK9 mRNA from the transgene
was not significantly different between WT-PCSK9 and
D374Y-low mice and was comparable to the level of endogenous mouse pcsk9 mRNA in whole-tissue extracts of both
the liver and distal small intestine, suggesting that human
PCSK9 was expressed under physiological control in these
mice. As expected, a significantly greater level of D374Y
Herbert et al
PCSK9 mRNA (normalised to GAPDH)
6.0
endogenous pcsk9
transgene PCSK9
4.0
2.0
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Control
WT PCSK9
D374Y
PCSK9low
D374Y
PCSK9high
Figure 1. Comparison of endogenous Pcsk9 and transgenic
PCSK9 mRNA levels in the livers of transgenic mice. Endogenous Pcsk9 or the transgenic PCSK9 mRNA was measured by
quantitative RT-PCR and expressed relative to the housekeeping gene Gapdh. Data are given as the mean⫾SD (n⫽3 mice
aged 20 weeks in 2 independent experiments).
transgene mRNA was detected in D374Y-high mice
(P⬍0.01). Analysis of mRNA with 2 alternative housekeeping genes (Ywhaz and Atp5b) gave similar results (data not
shown). Endogenous pcsk9 mRNA could also be detected in
the small intestine of control mice (⫺BAC) and mice expressing WT or D374Y PCSK9; however, expression was
more variable and at a lower level than in the liver relative to
gapdh; transgene mRNA was expressed at a similar level to
the endogenous mRNA in WT mice and was undetectable in
control mice (supplemental Table II). Despite the presence of
PCSK9 mRNA in the intestine, immunoblotting of different
tissues with an antibody to the c-myc tag on the human
PCSK9 transgene showed that the protein was expressed
almost exclusively in the liver, although some myc-PCSK9
protein was detectable in other tissues of D374Y-high mice
(supplemental Figure IIIA). We were unable to detect endogenous pcsk9 protein in any tissues using 3 commercially
available antibodies. As expected, mature myc-tagged
PCSK9 protein could be detected in the serum of the
transgenic mice (supplemental Figure IIIC). When PCSK9
protein levels were compared in liver membrane preparations
from chow-fed mice, there was no significant difference
between the WT-PCSK9 and the D374Y-low mice; however,
much higher levels were found in D374Y-high mice (supplemental Figure IIIB).
Expression of D374Y PCSK9 Has a Greater
Effect on Serum Lipids and Lipoproteins
Than WT PCSK9
To examine the effect of transgenic expression of PCSK9 on
serum cholesterol in chow- or cholesterol-fed animals, we
measured cholesterol levels in control and transgenic lines at
D374Y PCSK9 Transgenic Mice
1335
different points. The presence of 1 copy of human WT
PCSK9 resulted in a significant 2-fold increase in serum
cholesterol level from an early age (2.7⫾0.7 versus
5.5⫾1.3 mmol/L at the age of 4 weeks; P⬍0.001; n⫽15), and
this difference between control and WT-PCSK9 mice remained constant with time (3.0⫾0.7 versus 5.3⫾1.0 at the
age of 32 weeks; P⬍0.001; n⫽15). In contrast, serum
cholesterol levels at the age of 4 weeks were initially greater
in both D374Y-low mice (6.2⫾0.9 mmol/L) and D374Y-high
mice (7.3⫾0.9 mmol/L) compared with WT-PCSK9 mice
(n⫽15; P⫽⬍0.05) and continued to increase in both with
increasing age (8.2⫾1.6 and 8.4⫾1.9 mmol/L at the age of 32
weeks) (supplemental Table III). There was no significant
difference in serum cholesterol level between D374Y-high
and D374-low mice that received a chow diet. Serum
triglyceride levels, measured in 16- to 20-week-old mice
that had fasted overnight were significantly higher in mice
expressing D374Y PCSK9 at either a low copy number
(3.1⫾0.8 mmol/L; n⫽5; P⬍0.001) or a high copy number
(3.5⫾0.2 mmol/L; n⫽5; P⫽0.004) than in those expressing WT
PCSK9 (1.9⫾0.4 mmol/L; n⫽5) or in control nontransgenic
littermates (1.9⫾0.4 mmol/L; n⫽5). The expression of WT
PCSK9 did not affect serum triglycerides.
When the mice were fed a chow diet supplemented with
1% cholesterol (HCHOL) for 4 weeks (Table), serum cholesterol levels increased approximately 2-fold in WT-PCSK9
mice, 3-fold in D374Y-low mice, and 4-fold in D374Y-high
mice; however, there was no significant increase in the serum
cholesterol level of BAC-negative controls. As in chow-fed
animals, serum cholesterol levels in HCHOL-fed WT-PCSK9
mice then remained constant at 9.6⫾1.6 mmol/L during the
next 15 weeks; both D374Y lines showed an increase in
serum cholesterol levels during that period, reaching
23.1⫾7.0 mmol/L in D374Y-low and 32.6⫾7.4 mmol/L in
D374Y-high mice.
Feeding a High-Cholesterol Diet Reduces
PCSK9 Expression
To determine whether feeding a cholesterol-rich diet affected
transgene expression, we first measured liver cholesterol
levels in mice receiving a chow or HCHOL diet. Cholesterol
feeding increased liver total cholesterol level in all mice
(supplemental Figure IV), although the increase did not reach
statistical significance in D374Y-low mice, probably because
liver cholesterol levels were already increased in chow-fed
animals. We then measured PCSK9 mRNA levels. Both
endogenous and transgene mRNA levels were slightly, but
significantly, reduced in all animals fed the HCHOL diet
(supplemental Table IV); the reduction was greatest in
D374Y mice (2- to 3-fold), with a reduction of 1.5- to
2.0-fold in WT and D374Y-low mice. We then compared
myc-PCSK9 protein levels in the livers of chow- and cholesterol-fed mice. The results showed that there was no significant effect of the HCHOL diet on the amount of myc-tagged
PCSK9 protein in the livers of any of the mice lines
(supplemental Figure V).
D374Y PCSK9 Reduces, but Does Not Completely
Eradicate, LDLR Protein
Earlier studies have implicated a decrease in LDLR protein
levels in the mechanism of action of PCSK9, and adenoviral-
1336
Arterioscler Thromb Vasc Biol
Table.
July 2010
Serum Cholesterol Levels in Chow- and Cholesterol-Fed Mice*
Time Receiving Only
a Chow Diet
Genotype
4 wk
8 wk
Control†
2.4⫾0.6
WT
4.4⫾0.9
D374Y PCSK9
Low copies
High copies
Time Receiving a Chow Diet
Plus a 1% Cholesterol Diet
4 wk
8 wk
15 wk
2.7⫾0.9
2.7⫾0.9
3.1⫾1.2
3.1⫾1.5
4.9⫾0.6
9.3⫾1.8‡
9.0⫾1.6‡
9.6⫾1.6‡
6.5⫾1.8
6.6⫾1.0
17.6⫾4.7§¶
22.4⫾6.0§¶
23.1⫾7.0§¶
7.1⫾2.0
6.4⫾1.4
24.5⫾8.6§¶㛳
28.8⫾7.4§¶㛳
32.6⫾7.4§¶㛳
PCSK9 indicates proprotein convertase subtilisin/kexin type 9; WT, wild type.
*Data are given as mean⫾SD serum cholesterol level (measured in mmol/L) in mice fed only a chow diet for the
first 8 weeks of life, followed by mice fed a chow diet plus a 1% cholesterol diet for an additional 4 to 15 weeks.
†Non– bacterial artificial chromosome littermates.
‡P⬍0.05.
§P⬍0.001vs non-BAC littermate controls.
¶P⬍0.01 for D374Y mice vs WT.
㛳P⬍0.05 for D374Y mice (low vs high).
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mediated overexpression of PCSK9 in mice resulted in
undetectable LDLRs.6 To determine whether this was the
case in our transgenic mice expressing PCSK9 at more
physiological levels, we assayed hepatic LDLR protein by
semiquantitative immunoblotting of membrane fractions.
WT-PCSK9 and D374Y-low mice fed a chow diet contained
less hepatic LDLR protein than controls (Figure 2), with
lower levels present in D374Y-high mice. However, LDLR
protein was still clearly detectable, even in D374Y-high mice.
Feeding the HCHOL diet slightly increased hepatic LDLR
protein, as previously shown in cholesterol-fed rats.19
The Cholesterol Diet Induced a More Atherogenic
Lipid Profile in D374Y Mice
When fed a chow diet, the 3 transgenic mouse lines showed
virtually identical lipid profiles, with a slight increase in total
cholesterol in the apoB-containing LDL fraction compared
with their BAC-negative littermates (Figure 3A).
When fed an HCHOL diet, the lipid profile for control
mice remained unchanged, as expected20; there was an
approximately 2-fold increase in the LDL peak of WTPCSK9 mice (Figure 3B). However, the HCHOL diet induced
a more atherogenic lipid profile in both D374Y lines, with
large increases in apoB- and apoE-containing lipoproteins.
Figure 2. Immunoblotting of the LDLR protein in the livers of
transgenic PCSK9 mice. Liver membrane protein from transgenic
male mice aged 23 weeks, fed a chow diet or a high-cholesterol
diet for 15 weeks, as indicated on the figure, were separated on a
10% nonreducing gel and analyzed by immunoblotting with goat
anti-LDLR, detected with 680 nm– conjugated anti– goat IgG. Blots
are shown for 2 mice of each genotype and diet; similar results
were obtained in 3 independent experiments. PDI indicates protein
disulphide isomerase.
Total cholesterol appeared to be equally distributed between
the fractions containing large particles of the size of verylow-density lipoprotein and those containing LDL in the
D374Y-low mice; however, the total cholesterol level was
predominantly associated with the large fraction in the
D374Y-high mice. The cholesterol level in the HDL fraction
remained unchanged in all 4 lines.
Increased Secretion of ApoB-Containing
Lipoproteins in D374Y Mice
Immunoblotting of whole serum showed that serum apoB
levels were slightly increased in WT-PCSK9 mice compared
with control mice; however, these levels were significantly
increased in D374Y mice (Figure 4A). To determine whether
changes in the secretion of apoB-containing lipoprotein play
a role in hypercholesterolemia in PCSK9 transgenic mice, we
Figure 3. Lipoprotein profiles of PCSK9 transgenic mice fed a
chow or high-cholesterol diet. Whole serum from 1-year-old
mice fed a chow diet (A) or 23-week-old mice fed a high-cholesterol diet for 15 weeks (B) was fractionated by fast protein
liquid chromatography, and total cholesterol was assayed in
each fraction (n⫽5 for transgenic lines and n⫽3 for BACnegative controls).
Herbert et al
D374Y PCSK9 Transgenic Mice
1337
A
apoB100
apoB48
B
Serum triglyceride (mmol/l)
30
D374Y-low
ldlr-/PCSK9-WT
20
control
10
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0
20
40
60
80
Time (min)
Figure 4. Secretion of triglyceride in PCSK9 transgenic and
LDLR⫺/⫺ mice. A, Immunoblots of apoB in whole serum from
control and transgenic mouse strains as indicated, showing
apoB100 and apoB48. B, Chow-fed 16- to 20-week-old control
mice, transgenic WT-PCSK9 and D374Y-low mice, and
LDLR⫺/⫺ mice that fasted overnight and then were injected with
tyloxapol. Triglyceride was measured in serial serum samples as
indicated. Rates of secretion were compared using the general
linear model (P⬍0.001 for D374Y mice vs WT PCSK9 or
LDLR⫺/⫺ mice).
measured the rate of triglyceride secretion in chow-fed mice
that fasted, in which the lipase inhibitor, tyloxapol (Triton
WR1339), was injected. There was no significant difference
in secretion rate between WT-PCSK9 mice and BACnegative littermates; however, there was a significantly increased rate of secretion of triglyceride seen in the D374Ylow mice (Figure 4B). There was also no significant
difference in the secretion rate between ldlr⫺/⫺, WT-PCSK9,
or control mice, suggesting that the increased secretion in
D374Y-low mice was not solely the result of lack of LDLR
activity. As shown in Figure 4A, the basal levels of apoB48
and apoB100 were so high in the D374Y mice that it was not
possible to determine by semiquantitative immunoblotting
whether apoB secretion was also increased. Our license did
not permit use of radioactive tracers. It has been shown that
antisense-mediated downregulation of PCSK9 induces apobec-1
expression.21 Because there was an apparent slight increase in
the ratio of apoB100 to apoB48 in mice expressing mutant
PCSK9, we measured hepatic mRNA levels of apobec-1 and
apobec-1 complementation factor (A1cf), an intrinsic component of the apoB mRNA editing complex22; we found no
significant or consistent differences in mice fed a chow or an
HCHOL diet (supplemental Table V).
Extensive Atherosclerotic Plaques Are Found Only
in D374Y Mice
To investigate the consequences of the atherogenic lipid
profile in the transgenic mice, aortas from HCHOL-fed mice
Figure 5. Aortic lesions in PCSK9 transgenic mice. Aortas from
transgenic PCSK9 mice and their BAC-negative littermates
(males aged 23 weeks) fed a high-cholesterol diet for 15 weeks
were perfused with PBS, formalin, and Sudan IV. Dissected aortas were opened longitudinally, and lesion area was quantified
with respect to total surface area (n⫽5 mice for each group).
Representative examples from each experimental group are
shown, and the mean⫾SD lesion area is listed. *P⬍0.001 for
D374Y-low or D374Y-high mice vs WT-PCSK9 or control mice.
were perfused with the lipid stain, Sudan IV. Neither control
nor WT-PCSK9 mice showed any evidence of lesion formation in their descending aortas or aortic branches after 15
weeks on an HCHOL diet, whereas all D374Y-high mice
showed many lesions throughout their aortas. D374Y-low
mice also showed extensive lesion formation in their aortic
branches but varied in the amount of lesions seen in the
descending aorta (Figure 5). Lesions could be detected in the
D374Y-low and the D374Y-high mice after 10 weeks of an
HCHOL diet; these lesions were detected to a lesser extent
and were mainly situated in the main aortic arch and branches
(data not shown). Small lesions were also detectable in the
aortic arches of chow-fed D374Y mice, but not in other mice,
after 1 year (data not shown). Thus, expression of a single
copy of WT PCSK9 did not induce atherosclerosis, even in
cholesterol-fed animals, whereas expression of a single copy
of D374Y PCSK9 was sufficient to induce an atherogenic
phenotype that resulted in extensive plaque formation after 15
weeks of the high-cholesterol diet or a chow diet in older
mice.
Discussion
In the present study, we produced transgenic mice that
express human WT PCSK9 or the D374Y gain-of-function
mutant from a BAC that should contain all endogenous
regulatory sequences. Indeed, the WT-PCSK9 and D374Ylow mice, both of which had a single additional copy of
PCSK9 compared with BAC-negative littermate controls,
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Arterioscler Thromb Vasc Biol
July 2010
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expressed PCSK9 mRNA at levels comparable to that of
endogenous pcsk9 and with similar tissue distribution, suggesting that expression was under endogenous control. As
expected, D374Y-high mice, with approximately 5 copies of
PCSK9, expressed significantly more PCSK9 mRNA than
either WT-PCSK9 or D374Y-low mice.
Both the pro- and mature PCSK9 protein could be detected
in the livers of all 3 transgenic lines, with only the mature
protein secreted into the bloodstream (supplemental Figure I).
We were unable to detect significant amounts of human
PCSK9 protein in any other tissues, confirming previous
reports that most circulating PCSK9 is derived from the
liver,23 although pcsk9 mRNA could also be detected in the
small intestine, as previously shown.24 All 3 transgenic lines
displayed a modest increase in serum cholesterol level and a
decrease in LDLR protein level compared with controls. The
changes were more marked in D374Y-high mice; however,
even in these mice, detectable levels of LDLR protein
remained. Two previous transgenic mice that express human
PCSK9 have been described; expression was not driven by
the endogenous promoter in either case. In one case, gene
expression was under the control of the albumin promoter and
resulted in high levels of expression in the kidney25; in the
other case, expression was under the control of the human
apoE promoter and resulted in marked overexpression in the
liver, apparently comparable to that seen in mice expressing
adenoviral constructs of PCSK9.11 In both models, and in
contrast to our observations, the researchers reported that
LDLR protein was essentially abolished in the livers of mice
expressing WT PCSK9.
The lipoprotein phenotype in our mice expressing WT or
mutant PCSK9 was relatively mild when the mice were fed a
chow diet; however, when this diet was supplemented with
1% cholesterol, hypercholesterolemia in the D374Y-low
and the D374Y-high mice became more severe. Thus, as is
the case with the ldlr⫺/⫺ mouse,20 the phenotype reflected
that seen in patients heterozygous for gain-of-function
PCSK9 mutations15 only when the mice were fed a
high-cholesterol diet.
PCSK9 mRNA is regulated by sterols because feeding
mice or rats a cholesterol-rich diet resulted in reduction of
hepatic pcsk9 mRNA.4,19 We observed a moderate reduction
in the expression of liver pcsk9 mRNA, both endogenous and
transgene, after 15 weeks of a diet containing 1% cholesterol.
The main question we wanted to address with these mice
was whether the hypercholesterolemic effects of D374Y
PCSK9 were mediated entirely through reduction in LDLR
protein levels. Our data suggest that there is some additional
mechanism involved. First, we noted that fasting serum
triglyceride levels in chow-fed animals were increased in
transgenic mice expressing the mutant but not WT PCSK9.
Two recent studies26,27 have indicated that human serum
triglyceride levels are correlated with circulating PCSK9
protein. We also found that patients with FH heterozygous for
PCSK9 D374Y had significantly higher fasting serum triglycerides than patients with FH and LDLR defects.15 Second,
secretion of triglyceride-rich lipoproteins was also increased
in these mice compared with mice expressing WT PCSK9
and, more important, compared with ldlr⫺/⫺ mice. These data
suggest that expression of D374Y PCSK9 at physiological
levels results in increased secretion of triglyceride-rich lipoproteins from the liver, which is in agreement with previous findings of increased apoB secretion in isolated hepatocytes expressing D374Y, but not WT PCSK9.14 There also
appeared to be an increase in the proportion of apoB100 to
apoB48 in mice expressing D374Y PCSK9. However, this
mechanism did not involve changes in the expression of the
apoB mRNA editing enzyme, apobec 1, although ablation of
PCSK9 expression previously resulted in increased expression of this enzyme.21 Interestingly, it has been reported that
PCSK9-knockout mice show reduced postprandial lipemia
associated with reduced secretion of apoB from the intestine,24 whereas isolated hepatocytes from PCSK9-null mice
secrete less apoB100.8
In conclusion, we produced an animal model of FH caused
by the dominant D374Y mutation in PCSK9 that appears to
mimic the human disorder caused by heterozygous inheritance of D374Y PCSK9 more accurately than others previously described. The D374Y-transgenic mice should prove
useful for testing whether new therapies are effective in
reducing atherosclerosis.
Sources of Funding
This study was supported by the Medical Research Council; and in
part by grant PG/04/085/17409 from the British Heart Foundation
(Dr Soutar).
Disclosures
None.
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Increased Secretion of Lipoproteins in Transgenic Mice Expressing Human D374Y PCSK9
Under Physiological Genetic Control
Bronwen Herbert, Dilipkumar Patel, Simon N. Waddington, Emily R. Eden, Alexandra
McAleenan, Xi-Ming Sun and Anne K. Soutar
Arterioscler Thromb Vasc Biol. 2010;30:1333-1339; originally published online May 6, 2010;
doi: 10.1161/ATVBAHA.110.204040
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Increased secretion of lipoproteins in transgenic mice expressing human
D374Y PCSK9 under physiological genetic control
Bronwen Herbert, Dilipkumar Patel, Simon Waddington,
Emily R. Eden, Alexandra McAleenan, Xi-Ming Sun
and Anne K. Soutar
Supplementary Information
Supplementary Methods
Generation of PCSK9 transgenic mice
Human wild type and D374Y myc2-tagged PCSK9 cDNA was introduced by homologous
recombination17 into the exon1-intron1 region of PCSK9 in a bacterial artificial
chromosome containing human PCSK9 (BAC RP11-894E21 in pBACe3.6). Full-length
cDNA for human PCSK9, either wild-type or carrying the D374Y mutation, was
constructed to contain a carboxy-terminal myc2 tag. The 3’ untranslated region (UTR) of
the cDNA was replaced with that of bovine growth hormone (BGH) to prevent incorrect
recombination between the 3’UTR in human PCSK9 cDNA and the same region in the
BAC. Before insertion of the PCSK9 cDNA, the BAC was first modified by homologous
recombination to remove a second coding gene (USP24).
Linearised constructs were injected into B6/CBA pronuclei. Transgenic founders and
their transgenic offspring were identified by amplification of a 1400 bp fragment of the
human PCSK9 cDNA sequence from genomic DNA with a forward primer located in
exon 7 of PCSK9 and reverse primer in BGH 3’UTR.
1
Mice were maintained on a 12-h light/dark cycle and fed RM1 chow pellets or RM1+1%
cholesterol (HChol) (Lillico, UK) ad libitum. The mice were fed HChol diet for up to
fifteen weeks beginning at eight weeks of age. All animal experiments were performed
under Home Office Licenses 70/6014 and 70/6906. Ldlr-/- mice were generously
provided by M. Botto and T. Malik Imperial College London).
Copy Number Analysis by Southern Blotting
Genomic DNA was analysed by Southern blotting as described previously 1 with a full
length cDNA probe to PCSK9 2 and a cDNA probe to daf1, kindly provided by M. Botto
and T. Malik. The blots were quantified with a Phosphorimager (STORM 820,
Amersham Pharmarcia Biotech.).
Immunoblotting
Harvested tissues were frozen immediately on dry ice. For preparation of lysates, tissues
were homogenized in 50mM Tris pH8, 4mM EDTA, 150mM NaCl, 2% (v/v) Triton X100, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS. For membrane preparation,
tissues were homogenized in 20mM Tris-HCl pH8, 0.15M NaCl, 1mM CaCl2.2H20,
1mM PMSF, pelleted by centrifugation at 240,000g for 1hr and solubilised in 200 µl of
50mM HEPES pH7.4, 100mM NaCl, 1mM PMSF, 10mM EDTA, 10mM EGTA, 10mM
N-ethylmalemide, 2.2% (v/v) DMSO, 1% (v/v) triton X100 and 0.5mM leupeptin as
described by Van Driel 3. Protein content was assayed with the Bio-Rad DC Protein
Assay system with bovine serum albumin as standard. Samples (1µl of serum or 5µl of
FPLC fractions) and protein extracts (30µg of protein) were subjected to SDS-PAGE on
reduced polyacrylamide gels (non-reduced for detection of the LDLR protein) and
transferred to nitrocellulose membranes. Blots were incubated at ambient temperature
with primary antibodies at the following dilutions: rabbit anti–c-myc (Santa Cruz
2
Biotechnology Inc.) 1:3000, 1h ; mouse anti–c-myc (Cell Signalling Technology)
1:2000, 1h; goat anti-mouse LDLR (R&D Systems) 1:1000, 1h; goat anti-ApoB (Santa
Cruz Biotechnology Inc.) 1:500, 2 hours. As indicated in the figure legends, blots were
then incubated for 1h at ambient temperature with the appropriate secondary antibodies.
Bound horseradish peroxidase–conjugated secondary antibodies (Dako) were visualized
by chemiluminescence (Amersham Biosciences), whilst bound fluorescently-labeled
second antibodies (680nm conjugated antibodies, Invitrogen; 800nm conjugated
antibodies, LI-COR Biosciences), were visualized using the LI-COR Odyssey Infrared
Imaging System (LI-COR Biosciences). To control for loading, blots of membrane
proteins were re-probed with anti-protein disulphide isomerase (PDI; Santa Cruz
Biotechnology Inc.).
RT-PCR and real-time quantitative PCR
Mouse tissues were homogenized in liquid nitrogen and total RNA was isolated with
RNA-Bee (Ambio) according to the manufacturer’s instructions and quantitated by A260
(Nanodrop); DNase-treated RNA was reverse transcribed (High-Capacity cDNA Reverse
Transcription kit, Applied Biosystems).
Real-time quantitative PCR was performed on an ABI 7500 Fast Real-Time PCR
detection system with TaqMan® Universal PCR Master Mix (Applied Biosystems), with
primer-probe sets specific for either mouse or human PCSK9 (Applied Biosystems), and
mouse housekeeping genes Gapdh, Ywhaz and Atp5b (GeNorm) according to GeNorm
protocols (Supplementary Table I). Ct values were compared to a standard curve and
normalized to Gapdh, Ywhaz or Atp5b.
FPLC and analysis of lipoprotein distribution
3
Serum (50µl) was fractionated by fast-performance liquid chromatography (FPLC;
Pharmacia Smart System®) on a Superose 6 PC 3.2/30 column (GE Healthcare)19 with a
constant flow of 30 µl/min; 35µl fractions were collected and total cholesterol was
assayed in 5µl aliquots (Infinity Cholesterol kit, Microgenics).
Measurement of hepatic cholesterol levels
Lipids from ~30mg liver tissue were extracted with chloroform:isopropanol:triton X-100
(7:11:0.1 by vol), solubilised in 200µl of reaction buffer from the Cholesterol/Cholesteryl
Ester Quantification Kit (Calbiochem) and assayed for total, free and esterified
cholesterol, all according to the manufacturer’s instructions.
Analysis of atherosclerotic lesions
Aortic atherosclerosis was assessed as described21. Briefly, hearts were perfused
sequentially with PBS, 2% (v/v) formalin (Sigma) and Sudan IV solution (0.5% w/v
Sudan IV in 35% ethanol, 50% acetone, Sigma). Aortae were sectioned longitudinally
for en face imaging (Leica DC 500 imaging system). Lesions were expressed as a
percentage of the surface area of the aorta (ImagePro software, Media Cybernetics,
Bethesda, Md).
4
Supplementary References
1.
Sun XM, Webb JC, Gudnason V, Humphries S, Seed M, Thompson GR, Knight
BL, Soutar AK. Characterization of deletions in the LDL receptor gene in patients
with familial hypercholesterolemia in the United Kingdom. Arterioscler Thromb.
1992;12:762-770.
2.
Sun XM, Eden ER, Tosi I, Neuwirth CK, Wile D, Naoumova RP, Soutar AK.
Evidence for effect of mutant PCSK9 on apolipoprotein B secretion as the cause
of unusually severe dominant hypercholesterolaemia. Hum Mol Genet.
2005;14:1161-1169.
3.
van Driel IR, Davis CG, Goldstein JL, Brown MS. Self-association of the low
density lipoprotein receptor mediated by the cytoplasmic domain. J Biol Chem.
1987;262:16127-16134.
5
Supplementary Figures
Supplementary Figure I. Diagram showing the construction of the modified BAC
expressing human PCSK9.
The wide grey boxes represent human genes present in the BAC, with arrows indicating
direction of the coding sequence, the narrow grey box indicated intergenic human DNA
and the black lines, the BAC ends; PCSK9 and USP24 were full length genes; exon 1 was
absent from BSND. Recombination events in EL350 cells were selected with
spectinomycin (Sp) and the Spr gene was subsequently excised by activation of CRE
recombinase.
Supplementary Figure II. Detection of the transgene by PCR and Southern blotting
of genomic DNA from transgenic mice.
A. Genomic DNA from different female mice as indicated below the figure was
amplified with primers located in the BGH 3’-untranslated region and exon 7 of human
PCSK9. B. Genomic DNA from mouse liver (11 µg) was digested with Nhe1 and
analysed by Southern blotting. Blots were probed sequentially with a 1.1kb 32P-labelled
cDNA probe encompassing exons 1-11 of human PCSK9, and with a 501bp 32P-labelled
cDNA probe to the single-copy gene Decay Accelerating Factor (Daf), and developed
with a Phosphorimager. Transgene copy number was calculated as the ratio of
PCSK9/Daf, taking into account probe size.
Supplementary Figure III. Detection of transgenic PCSK9 protein in tissue lysates.
A. Protein tissue lysates (as indicated above the gel) from control or transgenic mice (as
indicated on the right), were separated on a 10% reducing gel and immunoblotted with
anti-myc to detect the transgenic myc-tagged PCSK9 protein, followed by HRP
6
conjugated anti-mouse IgG secondary antibody. All gels were analysed together, except
for D374Y-low mice. B. Liver membrane samples from chow-fed D374Y-low, WT and
D374Y-high mice were immunoblotted with mouse anti-myc (for tagged human PCSK9),
detected with 680nm conjugated anti-mouse IgG, and then rabbit anti-PDI, detected with
800nm conjugated anti-rabbit IgG and the Licor system, which provided a linear response
for all samples. C. Whole serum (1µl) from control or transgenic mice, as indicated
above the blot, was immunoblotted with anti-myc to detect PCSK9 protein.
(+HEK293), lysate of HEK293 cells transfected with human PCSK9 cDNA; P,
proprotein; M, mature PCSK9; PDI, protein disulphide isomerase as a loading control.
Supplementary Figure IV. Effect of the high cholesterol diet on hepatic cholesterol
content.
Total cholesterol was measured in the livers of control and transgenic mice fed chow or
HCHOL diet for 15 weeks. Lipids from liver tissue were extracted and assayed for total
cholesterol as described in the method. The values are mean ± standard deviation and
three mice for each group (P<0.05:HCHOL vs chow in control, D374Y mice; P<0.05:
WT mice compared to controls).
Supplementary Figure V. Comparison of PCSK9 protein in the livers of chow and
high cholesterol fed transgenic PCSK9 mice.
Liver membranes from chow- and cholesterol-fed transgenic mice as indicated on the left
were immunoblotted for myc-tagged PCSK9 protein and PDI as described in the legend
to Supplementary Figure 3. At least two independent experiments were carried out with
mice on each diet, with n = 3-5 mice for each. P, proprotein, M, mature PCSK9
7
Supplementary Table I. Sequences of oligonucleotide primers
Use
Primer or Probe
Sequence
RT-PCR of human Forward
5’-CTAGTTGCCAGCCATCTGTTGT
PCSK9 (BAC)*
Reverse
5’-GGCACCTTCCAGGGTCAAG
FAM-labelled probe
5’-TGCCCCTCCCCCGTGCCT
Forward
5’-ATTGCGGGCAGGGTCA
Reverse
5’-CTGTGGAAGCGTGTCCCAT
FAM-labelled probe
5’-TACCCGACTTCAACAGTGTGCCGG
Mouse pcsk9*
Genomic PCR to Forward PCSK9 ex 7
5’-GCTCCCGAGGTCATCACAGTTG
detect transgene
5’-ggcggccgcCATGCCTGCTATTGTCTT
Reverse BGH 3’-UTR
*Primers and probes designed with PE Applied Bio Systems software and purchased
from the same company. Primer/probe sets for mouse apobec1 (Mm00482894) and A1cf
(Mm 011948) were purchased from PE Applied Bio Systems Ltd.
Supplementary Table II. Endogenous and transgene PCSK9 intestinal mRNA
Genotype
Endogenous pcsk9
Transgene PCSK9
Control (-BAC) (n= 5)
0.68± 0.37
n.d.
WT (n= 5)
0.79± 0.31
0.51± 0.59
D374Y-high (n= 3)
0.79± 0.18
0.91± 0.45
Endogenous Pcsk9 or transgenic PCSK9 mRNA was measured by qRT-PCR in small
intestine of mice fed a chow diet, and expressed relative to the housekeeping gene
Gapdh.). Values are mean ± standard deviation; n.d. not detectable. Intestinal mRNA
was not available from D374Y-low mice
8
Supplementary Table III. Serum cholesterol levels in mice fed a chow diet.
Genotype
Serum cholesterol (mmol/l) at different ages (weeks)
4
8
12
16
20
24
28
32
Control
2.7±0.7
3.0±1.0
3.1±0.8
2.9±0.9
3.0±0.7
2.8±0.6
2.9±0.65
3.0±0.8
WT
5.5±1.3
5.9±1.2
5.8±1.0
5.4±1.19
5.4±1.0
5. 9±0.9
5.8±1.0
5.3±1.0
Low D374Y
6.2±0.9
6.4±1.3
6.1±0.9*
6.9±1.4*
7.0±1.3*
7.1±1.7*
7.4±1.4*
8.2±1.6*
High D374Y
7.3±0.8* 6.8±1.1* 7.6±1.5*
7.5±1.3*
7.5±1.5*
7.9±1.9*
7.8±1.4*
8.4±1.9*
Mice were weaned at three weeks of age and then fed a standard chow diet ad libitum. Serum cholesterol levels (mmol/l) were measured at four
weeks of age and then monthly. WT, mice expressing wild type PCSK9; Low PCSK9 and High PCSK9, mice expressing D374Y PCSK9 at high
or low copy number; control, non-BAC littermates. Values shown are mean ± standard deviation, n=15;
At each time point, all three transgenic mouse lines had significantly raised serum cholesterol levels (P<0.001) compared to the control group.
As confirmed by linear regression analysis, this increase was stable and maintained in WT mice (r2=0.041, P=0.63) but increased with age in
D374Y-low (r2=0.863, P<0.001) and D374Y-high (r2=0.0052, P<0.01) mice. Serum cholesterol levels were significantly increased in D374Yhigh at all time points and in D374Y-low mice from 12 weeks of age onwards in comparison to the WT mice (P<0.05).
9
Supplementary Table IV. Comparison of endogenous and transgene PCSK9
mRNA in chow and high cholesterol fed transgenic mouse livers.
mRNA level normalised to Gapdh
Endogenous Pcsk9
Chow
HCHOL
PCSK9 transgene
P*
Chow
HCHOL
P
n.d.
-
Control
1.77±0.18 0.96±0.32 0.04
n.d.
WT
1.66±0.38 0.94±0.22 0.009
1.72±0.57 0.71±0.21 0.0005
D374Y-low
1.74±0.12 0.92±0.19 0.001
0.86±0.27 0.52±0.22 0.044
D374Y-high 2.18±0.02 0.75±0.02 0.0001
4.95±1.53 2.33±1.66 0.017
Endogenous Pcsk9 or transgenic PCSK9 mRNA was measured by qRT-PCR in livers
of mice fed either chow or the HCHOL diet, and expressed relative to the
housekeeping gene Gapdh.). Values are mean ± standard deviation; n.d. not
detectable. P, significance HCHOL vs chow diet.
10
Supplementary Table V. Apobec1 and A1cf expression in chow and high
cholesterol fed transgenic mouse livers.
mRNA level normalised to Gapdh
apobec 1
Chow
A1cf
HCHOL
Chow
HCHOL
Control
1.37±0.06 0.68±0.34 0.99±0.28 1.68±1.23
WT
1.48±0.13 0.97±0.30 0.95±0.17 1.15±0.85
D374Y-low
1.22±0.03 0.60±0.55 2.39±0.42 1.56±0.72
D374Y-high 1.21±0.02 1.14±0.19 1.35±0.49 1.12±0.29
Endogenous apobec1 or A1cf mRNA was measured by qRT-PCR in livers of mice fed
either chow or HCHOL diet, and expressed relative to the housekeeping gene Gapdh.
Values are mean ± standard deviation, n=4 or 5.
11