From the Curriculum in Genetics and Molecular Biology
and the ¶ Department of Pathology and Laboratory Medicine,
University of North Carolina,
Chapel Hill, North Carolina 27599-7525
Received for publication, October 16, 2000, and in revised form, November 13, 2000
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ABSTRACT |
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The primary receptor mediating clearance of
apolipoprotein (apo)E- and apoB100-containing lipoproteins from the
circulation is the low density lipoprotein (LDL) receptor. Reduced
expression of the LDLR is believed to be a precipitating
factor in the pathogenesis of type III hyperlipoproteinemia (HLP) in
some humans homozygous for the apoE2 allele (APOE*2). To
test the effect of genetic changes in LDL receptor expression on the
pathogenesis of type III HLP, we have generated a variant allele at the
endogenous mouse Ldlr locus that expresses the human LDL
receptor transcript. Transcription of the human LDLR
minigene is regulated by the endogenous mouse promoter sequence, but a
truncation of 3'-untranslated region results in increased mRNA
stability. Consequently, in liver of heterozygotes, steady state levels
of mouse and human LDLR transcripts are 50 and 180% the
levels of total transcript in wild type mice, respectively. Overall,
the 2.3-fold normal level of LDLR message in heterozygotes
completely ameliorates type III HLP caused by the homozygosity for the
human APOE*2 allele, normalizing their plasma lipoprotein
profile. We conclude that a modest increase in expression of the
LDLR through message stabilization is sufficient to prevent
precipitation of type III HLP in mice.
Type III hyperlipoproteinemia (type III
HLP)1 is a disorder of
lipoprotein metabolism that leads to elevated plasma cholesterol and
triglyceride concentrations due mainly to an increase of
apoB-containing remnant lipoproteins. These particles are the product
of lipolytic processing of chylomicrons and very low density
lipoproteins (VLDL) derived from the intestine and liver, respectively.
Like low density lipoproteins (LDL), these are atherogenic
lipoproteins, and subjects with type III hyperlipoproteinemia are
predisposed to atherosclerosis and premature death from myocardial
infarction (1).
Type III HLP is a genetic disease associated with the expression of a
metabolically impaired apoE protein (2, 3) or apoE deficiency (4). It
is most commonly associated with individuals homozygous for the APOE*2
allele, the product of which has decreased affinity for the LDL
receptor compared with other common isoforms. However, homozygosity of
APOE*2 is necessary but not sufficient for the common form of type III
hyperlipoproteinemia, as only 5-10% of adult homozygotes develop this
disorder. In fact, most APOE*2 homozygotes have mild
hypocholesterolemia and reduced atherosclerosis risk (5).
It has long been appreciated that other genetic and environmental
factors besides possession of two APOE*2 alleles are
required for development of type III HLP. Type III HLP rarely manifests before adulthood, is more prevalent in men than women, and has an
earlier age of onset in men than women (6). Women tend to express type
III hyperlipoproteinemia only after menopause. Earlier onset is also
associated with obesity, excessive alcohol consumption, diabetes
mellitus, and hypothyroidism (1). The mechanism by which these
conditions induce type III HLP is unclear. One possibility may be that
these conditions result in down-regulation of hepatic LDL receptor
activity, leading to reduced uptake of atherogenic lipoproteins
containing apoE from plasma. For instance, estrogen has been shown to
increase hepatic LDL receptor activity (7). The decrease in plasma
estrogen levels in postmenopausal women would therefore be thought to
lead to decreased hepatic LDL receptor activity and increased
circulation time of remnant lipoproteins.
Previously we have generated mice that express human apoE2 in a
physiologically regulated manner by replacing the coding sequences of
the endogenous mouse Apoe gene with the human
APOE*2 allele (Apoe2/2 mice). These
mice exhibit full penetrance of the type III HLP phenotype, regardless
of age or gender in the presence of normal murine LDL receptor
expression (8). This suggested to us that genetic factors that trigger
the type III HLP phenotype in some humans are already present in mice.
One possible factor could be a relatively low hepatic level of the LDL
receptor in mice. Others have developed transgenic mice expressing
human apoE2 that exhibit features of type III HLP when endogenous apoE
is absent (9, 10). Unregulated overexpression of LDLR gene
by adenovirus-mediated gene transfer has been shown to be effective in
normalizing the plasma lipid profiles in these mutants (11). Here we
report that moderate and controlled overexpression of the LDL receptor completely ameliorates the type III hyperlipoproteinemia phenotype of
the Apoe2/2 mice. This increase in
LDLR expression was achieved through enhanced stability of
the LDLR mRNA in mice engineered to express human LDL
receptor in place of the mouse LDL receptor.
Construction of the Human LDLR Replacement Targeting
Vector--
As a 5' region of homology, a 7-kb fragment containing
intron 1 of the mouse Ldlr gene was isolated from plasmid
pSIT1. Plasmid pSIT1 was used for a targeted disruption of the murine
Ldlr gene by Ishibashi et al. (12) and was kindly
provided by Dr. Shun Ishibashi, University of Tokyo, Japan. A 12-kb
SalI/NotI fragment containing the human
LDLR minigene was isolated from plasmid pMY3, which was used
for generating LDLR transgenic mice by Yokode et al. (13) and was kindly provided by Dr. Masahiro Yokode,
University of Kyoto, Japan. The human minigene consists of a 7.5-kb
fragment of a SalI site in intron 1 through an
EcoRI site in exon 5 of the human genomic DNA, a 2.2-kb
fragment of an EcoRI site in exon 5 through a
SmaI site in exon 18 of the human LDLR cDNA
(14), and a 0.7-kb fragment of human growth hormone poly(A) addition signal sequence. As a 3' region of homology, a 1.4-kb fragment from a
SalI site in exon 4 through SacI site in intron 4 of the mouse gene was amplified from mouse genomic DNA by PCR using
primers designed from published cDNA sequence (15). These fragments were inserted into a TK Replacement of the Murine Ldlr Gene with the Truncated Human LDLR
Minigene--
The targeting vector was linearized and introduced into
embryonic stem cells, TC-1, and cultured under selection media as previously described (16). Cells resistant to both G418 and ganciclovir
were clonally isolated and screened by PCR using a Neo-specific primer 5'-GCT TCC TCG TGC TTT ACG GT-3' for the
targeting construct and a primer 5'-GCA AGA TGG CTC AGC AAG CA-3'
corresponding to intron 4 sequence. Correct targeting was confirmed by
Southern blot analysis using a probe derived from exon 4 of the mouse gene.
Chimeras were generated from targeted ES cells and bred with C57BL/6
mice to obtain germ line transmission of the modified chromosome. The
genotype of the modified allele (h) in the animals was determined by
the presence of a 300-bp PCR fragment produced by using the
Neo-specific primer above and a 3' exon 4-specific primer, 5'-GCA GTG CTC CTC ATC TGA C-3'. The wild type mouse
Ldlr allele (+) was detected as a 380-bp PCR fragment
produced by a 5' exon 4-specific primer 5'-CTC CCA GGA TGA CTT CCG
AT-3' and the 3' exon 4-specific primer above.
Mice heterozygous for the targeted Ldlr gene
(Ldlrh/+) were bred with
Apoe2/2 mice that were homozygous for targeted
replacement of the mouse Apoe gene with the human
APOE*2 gene (8). Doubly heterozygous mice were crossed to
Apoe2/2 mice again to generate mice homozygous
for the human APOE*2 gene and heterozygous for the targeted
Ldlr locus (Apoe2/2
Ldlrh/+ mice). These mice were further
crossed to Apoe2/2 mice to generate littermates
homozygous for the human APOE*2 gene with wild type mouse
Ldlr (Apoe2/2
Ldlr+/+ mice) or homozygous for the human
APOE*2 gene and heterozygous for the human LDLR
gene (Apoe2/2
Ldlrh/+ mice) for characterization. All
mice used for characterization were a mix of strains 129 and C57BL6/J
and were fasted for 4 h before plasma lipid analysis. Mice were
fed normal chow (Prolab RMH 3000, number 5P76, St. Louis, MO) or
western-style diet (0.15% (w/w) cholesterol and 21% (w/w) fat, TK
88137, Teklad Premier, Madison, WI) ad libitum.
Hepatic LDLR mRNA Analysis--
Mice were sacrificed with an
overdose of 2-2-2 tribromoethanol, and livers were harvested, flash
frozen in liquid nitrogen, and stored at Plasma Lipid Analysis--
Plasma was isolated and total
cholesterol, HDL cholesterol, and triglycerides were measured as
described previously (19). Equal volumes of plasma from at least five
sex- and age-matched animals were pooled, and lipoproteins were
separated by ultracentrifugation or by fast protein liquid
chromatography (FPLC) as described (20). Lipoprotein fractions were
dialyzed against phosphate-buffered saline, pH 7.4, and subjected to
SDS-PAGE analysis as described (20).
In Vivo Clearance of VLDL and LDL--
VLDL and LDL fractions
were isolated from plasma of apoE-deficient mice (21) by
ultracentrifugation for use as tracers for the turnover studies. The
VLDL and LDL were radioiodinated with 125I and
131I, respectively, using previously published procedures
(22, 23). The external jugular vein of recipient mice was exposed and
cannulated with a sterile tapered microrenathane catheter, and 3 µCi
(specific activity ~105 cpm/µg protein) of each
radiolabeled ligand were injected. Immediately after dose injection,
the catheter was removed, and the skin incision was closed using
interrupted sutures. Blood samples were collected by retro-orbital
sampling of anesthetized mice at various time points, and radiolabel
remaining in the plasma was quantified with a gamma counter.
Replacement of the Mouse Ldlr Gene with the Truncated Human LDLR
Minigene--
The targeting strategy used to disrupt the mouse
Ldlr gene and replace it with exons 2-18 of the truncated
human LDLR minigene is illustrated in Fig.
1. Homologous recombination between the endogenous locus (Fig. 1A) and the targeting construct (Fig.
1B) results in a hybrid gene in which the human
LDLR minigene is expressed under the control of the
endogenous promoter (Fig. 1C). All 5'-regulatory sequences
and exon 1 of the endogenous mouse locus are intact, but ~8 kb of
mouse DNA spanning 3' of intron 1 through exon 4 is replaced with the
human LDLR minigene. The 3'-UTR of the minigene is shortened
by a deletion of two of the three 3' "AU-rich elements" that
destabilize the mRNA transcript (24, 25). In addition, the human
LDLR minigene contains the poly(A) addition signal sequences of the human growth hormone gene. Because exon 1 codes only for the
signal peptide sequence, the mature protein transcribed from the
chimeric gene is entirely human protein. The modified locus was
transmitted to the F1 generation from chimeras that were made from one
of the targeted ES cell lines. All F1 heterozygote matings produced
normal litter sizes with a normal Mendelian segregation pattern of the
modified locus. We designate the modified allele as hLDLR
(or h) to distinguish it from the wild type mouse Ldlr allele (or +).
Increased Steady State LDLR mRNA Levels by Enhanced Message
Stability in Heterozygous Mice--
The expression of the
LDLR was confirmed by Northern blot analysis of mRNA
with a probe specific for the human LDLR mRNA. The human
message of ~3 kb was present only in animals heterozygous for the
hLDLR (Ldlrh/+) with no band
detected in wild type mice (Ldlr+/+) (Fig.
2A, upper band). The presence
of a 4.5-kb mouse Ldlr message was confirmed in both
Ldlr+/+ and
Ldlrh/+ mice by hybridizing the same
blot with a probe specific for the mouse message (Fig. 2A, lower
panel). The Ldlrh/+ mice had
~50% of wild type mouse Ldlr message levels, in
accordance with the loss of one copy of the mouse Ldlr
gene.
To estimate simultaneously the amount of both human and mouse messages
in homozygotes, we devised a primer extension strategy utilizing a
primer that hybridizes to a sequence in exon 3 shared in both the human
and mouse LDLR genes (Fig. 2B). The murine
message is extended 5 base pairs, and the human message is extended 3 base pairs before termination with dideoxy-CTP. In heterozygotes, the
steady state level of the human LDLR transcript is 3.8 ± 0.3 (n = 18) times the level of the mouse transcript
(p < 0.001). Since the levels of mouse Ldlr
message in the Ldlrh/+ mice are one-half
that in wild type mice, total message (mouse + human) is increased
2.3-fold (Fig. 2C). There is no down-regulation of the mouse
message in response to the higher levels of human message, nor
adjustment of total mRNA levels in the
Ldlrh/+ mice, despite the fact that
transcriptional regulatory elements in the LDLR gene are
intact. The steady state human message is reduced to 68% of the normal
chow level in response to dietary cholesterol loading (Fig.
2C) indicating that the sterol-responsive transcriptional
regulation of the receptor is intact and functional (26).
To determine if the increased steady state level of the
hLDLR mRNA is due to message stabilization, actinomycin
D and Reduction of Plasma Lipids in Mice Expressing hLDLR--
Both
heterozygous (Ldlrh/+) and homozygous
(Ldlrh/h) female mice expressing the
hLDLR exhibited approximately a 4-fold reduction in steady
state plasma cholesterol (mainly HDL cholesterol) when fed normal chow
(21 ± 3 mg/dl in Ldlrh/+,
n = 10; 28 ± 8 mg/dl in
Ldlrh/h, n = 8, compared with
85 ± 9 mg/dl in Ldlr+/+, n = 12 p < 0.00001). The reduction in plasma cholesterol
due to expression of the stabilized hLDLR transcript can be
viewed as a dominant trait, as there is little difference in plasma
cholesterol between heterozygotes and homozygotes for the
hLDLR. Consequently, all the remaining characterization was
with heterozygous mice and their wild type littermates. There was a
trend toward reduction of plasma triglycerides in
Ldlrh/+ mice (29 ± 6 mg/dl,
n = 10) compared with wild type mice (35 ± 7 mg/dl, n = 12), but this reduction was not significant
(p = 0.23). Both agarose gel electrophoresis (Fig.
4A) and FPLC analysis (Fig.
4B) of plasma showed patterns consistent with a reduction of
plasma lipid and HDL cholesterol. In the
Ldlrh/+ mice, both
When fed a diet containing 0.15% (w/w) cholesterol and 21% (w/w) fat,
plasma cholesterol levels in Ldlr+/+ and
Ldlrh/+ mice increased to 128 ± 10 and 81 ± 6 mg/dl, respectively. This increase was due mainly to
the increase in HDL cholesterol, as shown by FPLC analysis of plasma
from both groups of mice (Fig. 4D). This suggests that both
wild type and heterozygous mice respond similarly to dietary
cholesterol overload.
Increased LDLR Level Ameliorates Type III Hyperlipoproteinemia in
Apoe2/2 Ldlrh/+ Mice--
When a copy of the
hLDLR allele was introduced into the mice expressing human
APOE*2 in place of mouse Apoe, these mice
(Apoe2/2 Ldlrh/+)
exhibited a 2.5-fold increase in steady state total LDL receptor mRNA levels when compared with Apoe2/2 mice
(data not shown). The ratios between the mouse Ldlr and hLDLR mRNA levels were similar in
Apoe+/+ Ldlrh/+
and Apoe2/2
Ldlrh/+ mice.
The Apoe2/2 Ldlr+/+ mice
maintained on normal chow had type III HLP, with plasma cholesterol
levels of 268 ± 12 mg/dl and triglycerides of 157 ± 22 mg/dl. In contrast, their Apoe2/2
Ldlrh/+ littermates had normal
plasma cholesterol and triglyceride levels of 83 ± 4 and 40 ± 5 mg/dl, respectively. These values in the Apoe2/2 Ldlrh/+
mice are similar to those of wild type mice, suggesting that the
2.5-fold increase in LDLR expression is sufficient to
normalize the plasma lipids of the Apoe2/2 mice.
The marked reductions in plasma cholesterol and triglycerides observed
in the Apoe2/2
Ldlrh/+ mice can be accounted for by the
reduction in
FPLC analysis of plasma from the Apoe2/2
Ldlrh/+ mice (Fig. 5B)
confirmed that reductions in plasma cholesterol were due to the reduction of cholesterol in large lipoproteins (in the VLDL and IDL
range). There was no change in HDL cholesterol compared with Apoe2/2 Ldlr+/+ mice. In
addition, VLDL triglycerides are dramatically reduced in
Apoe2/2 Ldlrh/+
mice, whereas triglycerides in remnant particles (fractions 19-25) are
virtually eliminated. One diagnostic criterion for type III HLP is a
ratio of cholesterol/triglyceride in VLDL larger than 0.3. Although the
ratio in Apoe2/2 Ldlr+/+
mice is 0.67, the ratio in Apoe2/2
Ldlrh/+ mice is 0.2, less than the value
required for the diagnosis of type III HLP in humans.
The amelioration of type III HLP in the mice with modestly increased
LDL receptor levels is further shown by SDS-PAGE analysis of
apoproteins in lipoproteins isolated by ultracentrifugation (Fig.
5C). As previously reported, Apoe2/2
Ldlr+/+ mice fed normal chow have large amounts
of plasma apoB, especially apoB48, as well as apoE in the VLDL
fractions (8). In contrast, Apoe2/2
Ldlrh/+ mice have markedly reduced apoB
and apoE levels in all the non-HDL lipoprotein classes. One important
difference between mice expressing mouse apoE and mice expressing human
APOE*2 is that the Apoe2/2
Ldlrh/+ mice do not show any reduction
in apoAI (or cholesterol) in the HDL fractions, whereas
Apoe+/+ Ldlrh/+
mice have significant reductions in both.
When challenged by western style diet for 3 weeks,
Apoe2/2 Ldlrh/+
mice were still protected from type III HLP. Cholesterol levels increased in Apoe2/2
Ldlrh/+ mice (172 ± 43 mg/dl
n = 5) but were significantly lower than in
Apoe2/2 Ldlr+/+ mice
(546 ± 30 mg/dl, n = 3, p < 0.0001). The triglycerides in Apoe2/2
Ldlrh/+ mice did not increase compared
with levels on normal chow (35 ± 3 mg/dl n = 5),
whereas they doubled in the Apoe2/2
Ldlr+/+ mice (310 ± 84 mg/dl
n = 3). FPLC analysis of plasma from
Apoe2/2 Ldlrh/+
mice (Fig. 5D) fed a high cholesterol diet demonstrated the
continued protection against type III HLP, with increased cholesterol
predominantly in the HDL fractions. In contrast, the
Apoe2/2 Ldlr+/+ mice had
markedly increased VLDL triglyceride as well as VLDL-LDL cholesterol
with little change in HDL cholesterol (Fig. 5D). Taken together, these results demonstrate that a modest increase in the
expression of LDL receptor mRNA can ameliorate the type III HLP
phenotype in mice, making them more resistant to diet-induced hyperlipidemia.
Increased Clearance of Non-HDL Lipoproteins from Plasma of
Apoe2/2 Ldlrh/+ Mice--
To ascertain whether
the normalized plasma lipid and lipoprotein levels in
Apoe2/2 Ldlrh/+ mice are
due to an increase in functional LDL receptor activity, the clearance
of radiolabeled VLDL and LDL obtained from mice deficient in apoE was
measured. Fig. 6 shows that at 3 h
after injection, significantly more VLDL and LDL remain in the plasma of Apoe2/2 Ldlr+/+ mice
compared with Apoe2/2
Ldlrh/+ mice. This is consistent with
our previous observations that Apoe2/2 mice are
unable to clear VLDL from the plasma completely 4 h post-injection
(8). We conclude that the increased steady state LDLR
mRNA levels in Apoe2/2
Ldlrh/+ mice results in an increase in
functional LDL receptor activity, with an increased fractional
catabolic rate of VLDL, as well as LDL, and normalization of plasma
lipid levels.
In marked contrast to humans, in which type III HLP affects
5-10% of APOE*2 homozygotes, all mice homozygous for
targeted replacement of human APOE*2 exhibit
features of type III hyperlipoproteinemia regardless of age or gender
(8). In the current study, we have modestly increased steady state
hepatic LDLR mRNA levels by replacing the endogenous
mouse Ldlr gene with an hLDLR minigene with
increased mRNA stability. The Apoe2/2
Ldlrh/+ mice carrying this allele
exhibit a 2.5-fold increase in total hepatic LDLR message,
increased clearance of VLDL and LDL particles from the plasma, and a
normal plasma lipid phenotype. Not only is this modest increase in
hepatic LDLR mRNA sufficient to ameliorate the type III
hyperlipoproteinemia, the Apoe2/2
Ldlrh/+ mice are more resistant to
diet-induced hyperlipidemia than Apoe2/2
Ldlr+/+ mice.
Brown and Goldstein (26) have demonstrated a regulatory pathway that
results in the down-regulation of LDLR gene transcription in
response to increased intracellular sterol levels. However, regulation
of transcription is only one method by which steady state levels of
mRNA are altered. While the 5' regulatory elements of the human
LDLR have been studied in detail, the significance of 3'
regulatory elements in the regulation of cellular LDL receptor activity
is only beginning to be appreciated. A few studies of the
LDLR 3'-UTR indicate that it may play a significant role in regulation of LDLR mRNA levels. Wilson et al.
(28) have identified three AU-rich elements (AREs) at positions 2690, 3257, and 3438 of the human transcript (14) based on sequence homology
with the nonameric sequence UUAUUUAUU. This sequence is the minimal element contributing to a rapid turnover of several mRNAs such as
those encoding immediate early genes and cytokines (24, 25). By fusing
the 3'-UTR sequence of the human LDLR to the coding region
of human It is important to note that we found neither down-regulation of the
mouse gene accompanying the stabilized human gene nor normalization of
the total LDLR mRNA levels in heterozygotes. Furthermore, the messages for both mLDLR and
hLDLR alleles are reduced in response to increased dietary
cholesterol to similar degrees, maintaining the same steady state ratio
observed in mice fed normal chow. This suggests that there is no
feedback mechanism to adjust the LDLR mRNA levels
through transcriptional regulation when message stability is increased
in vivo.
Transgenic mice overproducing the human LDL receptor were reported
previously by Yokode et al. (13). These authors used the
mouse metallothionein-I promoter sequence to drive expression of the
human minigene in the transgenic mice. Similar to our
Ldlrh/+ mice, the transgenic mice on low
fat diet had markedly reduced levels of plasma cholesterol and
HDL cholesterol as well as apolipoprotein B and E. The most likely
explanation for the low levels of HDL cholesterol in mice with
increased LDL receptor activity is increased receptor-mediated
clearance of HDL particles with apoE. However, whether other mechanisms
such as inhibition of lipoprotein secretion by increased hepatic LDL
receptor activity (29) contribute to lower HDL cholesterol in these
mice require further studies. The transgenic mice generated by Yokode
et al. (13) are completely protected against diet-induced
hypercholesterolemia because of their unregulated overexpression of the
LDLR. In contrast, HDL cholesterol increased in
Ldlrh/+ mice in response to increased
dietary cholesterol, as in Ldlr+/+ mice. This
suggests the regulation of transcription of the LDLR gene by
increased cellular sterol levels in
Ldlrh/+ mice is intact.
Whereas many factors besides decreased LDL receptor activity may
precipitate type III HLP in human APOE*2 homozygotes (30), it is clear that high hepatic expression of the LDL receptor is sufficient to overcome this phenotype in mice. Previous studies that
test the effect of LDL receptor overexpression on type III HLP in mice
have used very high levels of uncontrolled overproduction of the LDL
receptor (9, 31). Our study is the first to show that as little as a
2.5-fold increase in LDL receptor mRNA results in increased
clearance of VLDL and a 3-4-fold reduction of plasma cholesterol and
triglycerides in mice expressing apoE2 fed normal chow. The fact that
most human APOE*2 homozygotes are slightly hypolipidemic,
but all Apoe2/2 Ldlr+/+
mice are hyperlipidemic, suggests a possibility that the set point of
expression of the LDLR may be higher in humans than in mice.
Murine apoE can efficiently mediate clearance of plasma lipoproteins by
LDL receptor-independent
mechanisms2 Thus, a
relatively low set point of LDLR expression may not
influence the overall metabolism of apoE-containing lipoproteins in
mice except when mouse apoE is replaced with human apoE2, which is far
less effective in mediating LDL receptor-dependent uptake of apoE-containing lipoproteins.
Three-quarters of LDL receptor proteins in
Apoe2/2 Ldlrh/+
heterozygotes are of human sequence. Our unpublished studies using
mouse fibroblast cells show that VLDL containing mouse apoE or human apoE3 or apoE4 isoforms bind to the mouse LDL receptor equally well,
whereas VLDL with apoE2 binds with similar affinity but with 50%
Bmax compared with VLDL containing the other
apoE isoforms.2 The results were similar when human cells
were used.2 Thus, species
differences in the affinity of apoE for the LDL receptor do not appear
to be playing a significant role. In contrast, Corsini et
al. (32) have shown that human LDL has much lower affinity to
mouse receptor than to human receptor, suggesting significant species
effects are present in the interaction between apoB100 and the LDL
receptor. Although we did not observe any increase of LDL in either
heterozygotes (Fig. 4) or homozygotes (data not shown), our present
study was carried out in Apoe2/2
Ldlrh/+ heterozygotes to reduce the
potential complexities induced by interactions between mouse LDL and
the human LDL receptor.
Several studies have shown that cellular LDL receptor activity can be
regulated by post-transcriptional mechanisms, including changes in
mRNA stability as well as altered protein stability. For example,
treatment of HepG2 cells with phorbol-12-myristate-13-acetate has been
shown to increase the stability of LDLR mRNA 2-2.5-fold by an indirect effect of destabilization of the actin cytoskeleton by
this agent (33). Sequences in the extreme 3'-UTR of the LDLR have been shown to confer association of this RNA with the actin cytoskeleton. Lack of this region inhibits the PMA-induced
stabilization of this RNA (28). Gemfibrozil, a fibrate drug used in the
treatment of type III hyperlipoproteinemia in humans, also has been
shown to enhance LDLR mRNA stability in human hepatoma
cells (34). In this study pravastatin, an inhibitor of
3-hydroxy-3-methylglutaryl-coenzyme A reductase, significantly
increased sterol-responsive element-dependent transcription
(with no effect on LDLR mRNA stability), whereas gemfibrozil increased LDLR mRNA stability 4-6-fold with
no change in transcription rate. Whereas both drugs have efficacy in
treating type III hyperlipoproteinemia, pravastatin lowers LDL
cholesterol preferentially, and gemfibrozil treatment results in larger
reductions in VLDL cholesterol and triglycerides (35). Whether these
differences are the result of different mechanisms leading to increased
LDL receptor activity or are due to other effects of these drugs on peripheral lipolysis or hepatic VLDL secretion has not been resolved.
That the regulation of LDLR mRNA stability may have a
physiological role is suggested by the fact that depletion of hepatic sterol by the inhibition of squalene synthase results in increased LDLR mRNA transcription, stability, and translation in
rats (36). Whether alterations of LDLR mRNA levels in
humans due to regulated alterations in mRNA stability can be a
"precipitating factor" for type III hyperlipoproteinemia is
unclear. Nevertheless, our present study makes clear that changes in
hepatic LDLR levels due solely to differences on
LDLR mRNA stability can have a profound effect on plasma
lipid levels in a mouse model of type III HLP.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Neo vector to yield the targeting construct shown in Fig. 1A below. The neomycin phosphotransferase
(Neo) gene was placed in the same transcriptional
orientation as the LDLR gene. The human minigene and the
Neo gene replaces ~8 kb of DNA from an XhoI
through a SalI site in exon 4 of the mouse Ldlr gene.
70 °C. RNA was prepared
from the tissue using Trizol reagent following standard protocols (17).
For Northern blot analysis, a 380-bp fragment of mouse exon 4 and a
350-bp fragment of human exon 4 were used as probes specific for the
mouse and human genes, respectively. A primer extension assay was
designed to quantitate both human and mouse message simultaneously
using a primer 5'-GGA GCA CGT CTT GGG GGG ACA GCC T-3' corresponding to
a shared sequence within exon 3 of the two LDLR genes.
Dideoxy-CTP terminates extension of the primer, resulting in murine
message extension by 5 bp and human message extension by 3 bp. The 30- and 28-bp fragments were separated in a denaturing 20% polyacrylamide gel. A primer 5'-GCA GCG AAC TTT ATT GAT GGT ATT 3' was included in
each reaction to determine the expression of the
glyceraldehyde-3-phosphate dehydrogenase (Gapdh) gene as an
internal control. Primer extension analysis was performed on 50 µg of
total mRNA as previously described (18). Image densitometry was
used to determine relative amounts of message (FLA-2000, Fuji Photo
Film USA Inc., Elmsford, NY). To determine the decay rate of the
LDLR mRNA, heterozygous animals were injected via tail
vein with actinomycin D (150 µg/100 g body weight) and
-amanitin
(50 µg/100 g body weight). Animals were sacrificed at various time
points after injection, and liver mRNA was prepared using standard
protocols for primer extension analysis.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Replacement of the murine Ldlr
gene with the human LDLR minigene.
A, genomic organization of the mouse Ldlr gene
containing exons 1-18 (black boxes). The three AU-rich
elements (ARES) in exon 18 are depicted as white
rectangles. An asterisk indicates the position of the
stop codon. B, the targeting construct containing the 5' and
3' arms of mouse homology (thick black lines) interrupted by
exons 2-4 of the human LDLR gene followed by the
LDLR cDNA starting with exon 5. White boxes
indicate exons and thin lines indicate introns of the human
gene. The exon 18 is truncated after the first ARE (white
rectangle) and followed by the human growth hormone poly(A)
addition signal sequence (black box). A
neomycin-phosphotransferase (Neo) and thymidine kinase gene
(TK) was inserted for selection of targeted ES cell
colonies. C, the correctly targeted locus results in a
chimeric gene encoding the human LDLR.
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[in a new window]
Fig. 2.
Expression of the LDLR
genes. A, upper panel, Northern blot
analysis using a probe specific for the human LDLR gene. RNA
was isolated from wild type (+/+) or
Ldlrh/+ (h/+) mice as
genotyped by PCR analysis. Arrows indicate positions for
28 S and 18 S RNA. Lower panel, the blot above was
stripped and re-hybridized to a probe specific for the mouse
Ldlr gene. B, primer extension analysis of
hepatic RNA isolated from wild type mice (+/+) and
Ldlrh/+ heterozygotes (h/+).
The bands representing the mouse LDLR, the human LDLR, and
Gapdh transcripts are indicated. C, liver RNA was
isolated from six female +/+ mice, six female h/+ mice fed normal chow,
and nine h/+ mice fed western style diet. Primer extension was
performed on individual samples distinguishing human and murine
transcripts, and each was quantified by densitometry and values are
shown as mean ± S.E. Mouse message, black box; human
message, white box. Both mouse and human LDLR messages are
significantly reduced (* p < 0.001) in mice fed
western-style diet compared with those in mice fed normal chow.
-amanitin were administered in vivo to
Ldlrh/+ mice. These drugs inhibit
transcription, allowing for the measurement of mRNA decay rates
(27). Differentiation between the mouse and human LDLR
messages by primer extension was then used to determine relative decay
rates of these transcripts in the liver (Fig.
3A). The time course analysis
(Fig. 3B) shows that the human message decayed with a
half-life of about 160 min while the mouse message decayed with a
half-life of 60 min. This confirms that loss of two AREs in the human
LDLR transcript results in a large increase in message
stability in vivo in the liver of mice.
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Fig. 3.
Increased human LDLR
mRNA stability. A, a radiograph of primer
extension analysis of liver RNA isolated from individual mice given a
bolus tail vein injection of actinomycin D (150 µg/100 g body weight)
and -amanitin (50 µg/100 g body weight) at time 0. Mice were
sacrificed at the times post-injection indicated. Each lane represents
the RNA from an individual Ldlrh/+
mouse. B, decay of human and mouse LDLR mRNA in
heterozygotes. mRNA levels for hLDLR (open circles) and
mLDLR (closed circles) are expressed relative to the average
levels of mLDLR at time 0. Mean ± S.E. from 3 to 6 animals at
each time point except for time 0 (10 animals). Half-life for hLDLR
(160 min) and for mLDLR (60 min) was calculated from the first 2 h
of the data.
- and
pre-
-migrating particles, as well as
-migrating particles, were
dramatically reduced. SDS-PAGE analysis of plasma lipoproteins isolated
by sequential density ultracentrifugation showed that the reduction in
plasma lipid was accompanied by reductions in plasma apoproteins B, E,
and AI (Fig. 4C). Although apoB100 is present in IDL-LDL
fractions (p = 1.04-1.06 g/ml) of the wild type mice,
it was virtually absent in the IDL-LDL fractions of the
Ldlrh/+ mice (the protein band
corresponding to apoB100 was slightly degraded in this example). Plasma
apoE was also reduced in all fractions of plasma from
Ldlrh/+ mice, with only a small amount
of apoE remaining in the largest, lowest density particles (VLDL in
fraction p = 1.006 g/ml).
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Fig. 4.
Plasma lipid and apolipoprotein distribution
in wild type (+/+) or heterozygote (h/+) mice. A,
fasted plasma (1 µl) from males fed normal chow was electrophoresed
in a 1% agarose gel and stained with Fat Red 7B. The positions of
-,
-, and pre-
-lipoproteins are indicated. B,
pooled plasma (100 µl) from 6 male mice fed normal chow was analyzed
by FPLC on a Superose 6B column. 0.5-ml fractions were collected, and
lipid was measured as µg/fraction. TC, total cholesterol
(open circles); TG, triglycerides (closed
circles). The numerical values represent the total
plasma levels in mg/dl ± S.D. from six animals. C,
pooled plasma (1 ml) from Apoe+/+
Ldlr+/+ (+/+). and
Apoe+/+ Ldlrh/+
(h/+) mice fed normal chow was fractionated by sequential density
ultracentrifugation, and each fraction was electrophoresed in a
denaturing 3-20% SDS-PAGE gradient gel and stained with Coomassie
Brilliant Blue. Density fractions are as follows: 1.006,
<1.006 g/ml; 1.02, 1.006-1.02 g/ml; 1.04, 1.02-1.04 g/ml; 1.06, 1.04-1.06 g/ml; 1.08,
1.06-1.08 g/ml; 1.10, 1.08-1.10 g/ml; and 1.21,
1.10-1.21 g/ml. D, 100 µl of plasma pooled from at least
five male mice fed western-style diet was analyzed by FPLC on a
Superose 6B column. 0.5-ml fractions were collected, and lipid was
measured as µg/fraction. TC, total cholesterol (open
circles); TG, triglycerides (closed
circles). The numerical values represent the total plasma levels
in mg/dl ± S.D.
-VLDL, as shown by agarose gel electrophoresis of whole
plasma (Fig. 5A). The lipid
content of
-migrating HDL particles in
Apoe2/2 Ldlrh/+
mice was not reduced in the presence of the hLDLR allele,
which is in distinct contrast to the marked reduction of HDL in
Apoe+/+ Ldlrh/+
mice, which have wild type mouse apoE protein.
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Fig. 5.
Plasma lipid and apolipoprotein distribution
in mice expressing human apoE2 without (2/2 +/+) and with a human
LDLR allele (2/2 h/+). A, fasted plasma (1 µl) from males fed normal chow was electrophoresed in a 1% agarose
gel and stained with Fat Red 7B. B, pooled plasma (100 µl)
from 6 male mice fed normal chow was analyzed by FPLC on a Superose 6B
column. 0.5-ml fractions were collected, and lipid was measured as
µg/fraction. TC, total cholesterol (open
circles); TG, triglycerides (closed
circles). The numerical values represent the total plasma levels
in mg/dl ± S.D. C, pooled plasma (1 ml) from
Apoe2/2 Ldlr+/+
(2/2 +/+) and Apoe2/2
Ldlrh/+ (2/2 h/+) mice fed
normal chow was fractionated by sequential density ultracentrifugation.
Each fraction was electrophoresed in a denaturing 3-20% SDS-PAGE
gradient gel and stained with Coomassie Brilliant Blue. Density
fractions are as follows: 1.006, <1.006 g/ml;
1.02, 1.006-1.02 g/ml; 1.04, 1.02-1.04 g/ml;
1.06, 1.04-1.06 g/ml; 1.08, 1.06-1.08 g/ml;
1.10, 1.08-1.10 g/ml; and 1.21, 1.10-1.21 g/ml.
D, 100 µl of plasma pooled from at least five male mice
fed western-style diet was analyzed by FPLC on a Superose 6B column.
0.5-ml fractions were collected, and lipid was measured as
µg/fraction. TC, total cholesterol (open
circles); TG, triglycerides (closed
circles). The numerical values represent the total
plasma levels in mg/dl ± S.D.
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Fig. 6.
Plasma VLDL and LDL clearance rates in mice
fed normal chow. VLDL and LDL were isolated from apoE-deficient
mice by ultracentrifugation. VLDL labeled with 125I and LDL
labeled with 131I were injected via jugular vein into
Apoe2/2 Ldlr+/+
(2/2 +/+) or Apoe2/2
Ldlrh/+ (2/2 h/+) mice. At
3 h post-injection, mice were bled retro-orbitally, and the
remaining radiolabel in plasma was measured. Values shown are
percentage of injected radiolabel amount remaining at 3 h in
mean ± S.D.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-globin gene, Wilson et al. (28) demonstrated that the 3'-UTR sequence of the LDLR gene confers a short
constitutive half-life to the otherwise stable
-globin transcript in
cultured cells. They showed that fusion constructs containing all three AREs have a 10-fold higher mRNA turnover rate compared with
constructs lacking all three AREs. The three AREs contributed in an
additive fashion to the mRNA destabilization rate, with constructs
containing only the 5'-most ARE having just a 3-fold increase in the
mRNA degradation rate. Since the chimeric transcript produced by
Ldlrh/+ mice contains only human
sequence up to nucleotide 2804, it lacks two of the AREs. The lack of
these two AREs results in a 3-fold increase in the stability of the
LDLR message in vitro (28), an increase in
stability similar to what is seen in the mice in vivo in our
current study. Thus in Ldlrh/+ mice
there was a 3.8-fold higher steady state level of chimeric human
message compared with wild type mouse message. Since both mLDLR and hLDLR alleles have identical
transcriptional regulatory sequences, the increase in steady state
levels of hLDLR message over the wild type mLDLR
message must be the direct result of this increase in message stability.
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ACKNOWLEDGEMENTS |
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We thank E. Avidervich and K. Kluckman for their excellent technical help; Drs. M. Yokode and S. Ishibashi for generously supplying plasmids used in generating the targeting construct; Drs. J. Parks and T. Smith for help in lipoprotein clearance experiments; and J. Knowles, Dr. O. Smithies, Dr. P. Sullivan, and Dr. S. Quarfordt for valuable discussions.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants HL42630 and GM20069.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.
§ Both authors contributed equally to this work.
To whom correspondence should be addressed: Rm. 701, Brinkhous-Bullitt Bldg., Dept. of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, NC 27599-7525. Tel.: 919-966-6914; Fax: 919-966-8800; E-mail: nobuyo@med.unc.edu.
Published, JBC Papers in Press, November 13, 2000, DOI 10.1074/jbc.M009423200
2 C. Knouff, V. Clavey, and N. Maeda, unpublished observations.
3 C. Knouff, V. Clavey, and N. Maeda, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: type III HLP, type III hyperlipoproteinemia; apoE, apolipoprotein E; FPLC, fast performance liquid chromatography; Gapdh, glycerophosphate dehydrogenase gene; HDL, high density lipoprotein; LDL, low density lipoprotein; LDLR, low density lipoprotein receptor; Neo, neomycin phosphotransferase gene; VLDL, very low density lipoprotein; bp, base pair; kb, kilobase pair; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; FPLC, fast protein liquid chromatography; AREs, AU-rich elements; IDL, intermediate density lipoprotein.
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