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https://www.abbs.info ISSN 0582-9879 |
Excessive
Expression of the Scavenger Receptor Class A Type I can Significantly Affect
the Serum Lipids
GAO Jun, LIU De-Pei*,
HUANG Yue, DONG Wen-Ji, WU Min, FENG Dong-Xiao, LIANG Chih-Chuan
( National Laboratory of Medical Molecular Biology, Institute of Basic Medical
Sciences,
Chinese
Academy of Medical Sciences & Peking Union Medical College, Beijing 100005,
China )
Abstract
Scavenger receptor (SR) is characterized by its
ability to bind negatively charged macromolecules, particularly the modified
lipoproteins that are pertinent to the development of vascular disease. To
determine the role of excessive scavenger receptor A in the serum lipoprotein
metabolism, transgenic mice lines with mouse scavenger receptor A gene type I (SR-AI) under the control of human SR-AI enhancer and
metallothionein gene promotor were established. After zinc induction, the
expression of SR-AI in transgenic mice was a little
higher than the controls, but the serum lipids levels were significantly
different from the controls, especially the cholesterol. These results
demonstrated that overexpression of SR-AI significantly affected the serum
lipids levels.
Key
words scavenger receptor (SR); metallothionein gene
promotor; transgenic mouse; excessive expression; serum lipids
Scavenger receptors (SRs) are an expanding family containing several
kinds of membrane receptors which are characterized by their ability to bind
negatively charged macromolecules, such as chemically modified or altered
molecules and, in particular, the modified lipoproteins that are pertinent to
the development of vascular diseases[1,2]. Scavenger receptor class
A (SR-A) is the first scavenger receptor that has been identified and the
molecular cloning of this receptor discloses 3 isoforms, SR-AI, SR-AII and
SR-AIII, which are the products of the alternative splicing of SR-A and different from each other in
the cysteine-rich domain[3,4]. SR-A is a trimeric membrane
glycoprotein consisting of six structural domains including the collagen-like
domain, which is involved in ligand binding[5,6]. SR-A is expressed
predominantly in macrophages and to a much lower extent in smooth muscle cells
and endothelial cells[7-9].
Although SR-A is associated with multiple activities, people still
pay much attention to its involvement in vascular pathology because of its
relationship to atherosclerosis. Several investigators reported that scavenger
receptor and its mRNA have been detected in foam cells in atherosclerotic
plaques in vivo[10,11]. Freeman et al. [12] demonstrated that transfection of cDNA of SR
to Chinese hamster ovary cells leads to their conversion to foam-like cells in
the presence of modified low density lipoprotein (LDL). The produced transgenic
mice indicate that the SR plays an important role in the foam cell formation in vitro and in vivo[13,14]. The mechanism has been well interpreted
that SRs mediate the uptake of modified low density lipoprotein by macrophage.
The accumulation of lipids via this process is thought to result in the foam
cell formation in developing atherosclerotic plaques.
Although SR-A is closely related with
atherosclerosis, it isn’t thought to affect the plasma lipids levels because
the modified LDL is much less than natural LDL in the plasma. So the change of
plasma lipids levels has nothing to do with SR-A. In fact, under different
genetic backgrounds, the effects of SR-A will be different. Suzuki et al.[15] demonstrated that the plasma
cholesterol levels were higher in apoE -/- SR-/- mice
compared with apoE -/- mice. Sakaguchi et al.[16] found that after 4 weeks on high-fat
diet, plasma cholesterol levels were 20% higher in the control LDLR-/- mice than in the SR-/-
LDLR-/- mice. However,
after 12 weeks on the high-fat diet, plasma lipids did not differ between the
two groups. Similarly, Winther et al.[17]
found that the serum cholesterol level of male SR-/- E3L
mice was 50% lower than SR-A+/+ E3L mice.
We hypothesized that overexpression of SR-A can affect the levels of
the lipids in the serum. To determine the role of excessive macrophage SR-A in
the plasma lipoprotein metabolism, we established several lines of transgenic
mice overexpressing the macrophage SR-A under the control of the
metallothionein promoter. In the present study, we demonstrated that after zinc
induction the expression of the transferred gene (transgene) increased
moderately while affected the serum lipids significantly.
1 Materials
and Methods
1.1 Plasmid construction
The constructs containing
the mouse SR-AI, hESR and metallothionein(MT)
promoter were generated. Initially the MT promotor fragment was
separated from the mMT-1 plasmid and inserted into the pGL3 basic plasmid. The
mouse macrophage scavenger receptor class A type I (mSR-AI) was
amplified by RT-PCR using the RNA taken from the mouse macrophage as a
templete. Then the mSR-AI fragment was inserted into the BglII site in the pGL3-MT plasmid. The hESR
was amplified by PCR using the human genomic DNA as templete and inserted into
the KpnI site of the pGL3-PMT–mSR-AI
plasmid. At last the fragment between HindIII
and XbaI was deleted and the plasmid
was self-ligated.
1.2 Generation and characterization of the
transgenic mice
Transgenic mice overexpressing the mouse gene SR-A were generated as previously described by oocyte
microinjection[18]. DNA of the construct of interest was
microinjected into the pronuclei of fertilized mouse eggs (from F1 females of
mouse strain Km×ICR) at a
concentration of 2 mg/L in injection buffer [ 10 mmol/L Tris (pH 7.4), 0.1
mmol/L EDTA, and 100 mmol/L NaCl ]. Embryos that survived from microinjection
were maintained for 30 min in M16 medium and then implanted into the oviduct of
pseudopregnant (mouse strain Km) F1 female mice. Transgenic founders were
identified via PCR and Southern blot of tail DNA. PCR primer pairs were as
follows: forward 5′-CGGGGACAAGGGCCCACTAAAAGA-3′ and reverse
5′-TCTGCAGGAGACAGCTGATCTTG-3′. Genomic DNA (10 μg) from F1 mice was used to
determine the copy number by quantitation of Southern blot using PhosphorImager
(Molecular Dynamic).
1.3 Animal treatment procedure
To induce expression of the transgene, animals were given water
containing 20 mmol/L ZnSO4 for 1 week. Fasting (12 h from 21:00 pm
of the first day to 9:00 am of the second day) blood was obtained by tail
bleeding. The concentrations of total cholesterol and triglycerides in the sera
were determined by enzymatic procedures.
1.4 RNA
assay
The total cellular RNA was isolated from
macrophages, which were taken from the mice’s abdominal cavity, using Trizol
reagent(Gibco BRL). The cDNAs were assayed by RT-PCR in a PCR system containing
2 μCi [α-32P]dCTP. Primer1 forward
5′-CGGGGACAAGGGCCCACTAAAAGA-3′, Primer 1 reverse 5′-TCTGCAGGAGACAGCTGATCTTG-3′;
Primer 2 forward 5′-TGACCTCAACTACATGGTC-3′, Primer 2 reverse
5′-CTGTTGCTGTAGCCGTATT-3′.
The total RNA taken from liver, spleen, heart, brain and
kidney were separated by electrophoresis through a denaturing agarose gel (1% W/V)
containing 7.5% formaldehyde and transferred to Hybond-N+ membrane
according to manufacturer’s recommendations. Blots were hybridized with a [α-32P]
labeled probe of mouse mSR cDNA.
1.5
Statistical analysis
Non-transgenic littermates were used as
control mice. Results from each group of samples (the serum lipids) were
reported as the mean + standard deviation, and statistical analyses of
the means were performed with the t-test. P<0.05
denoted statistical level of significance.
2. Results and Discussion
2.1 Plasmid construction and transgenic
mouse establishment
Macrophage scavenger receptors are trimeric proteins with tightly
conserved novel structure across a wide evolutionary expanse. The physiologic
role of these receptors remains uncertain, but their ability to bind diverse
ligands, particularly modified forms of LDL, implicates that they are important
in the process of atherosclerotic foam cell formation. In the previous study,
there was a conflicting relationship between mSR and serum lipids. In the
present study, we tried to make it clear whether the excessive expression of
mSR can affect the metabolism of the serum lipids.
For the sake of sepecific and excessive expression of the transgene,
we inserted the human scavenger enhancer hESR and
metallothionein promoter (PMT) into the injected DNA
construct. Then the constructed plasmid DNA was sequenced and proved to contain
the three parts of the transgene in the correct sequence (Fig. 1). Because
there was no appropriate enzymatic digestion site for linearization of the
plasmid, we designed two primers to amplify the whole transgene including the
metallothionein gene promoter, the human scavenger gene enhancer and the entire
mouse macrophage receptor cDNA. In order to avoid the default of the
amplification, we used pfu DNA
polymerase, a kind of high fidelity thermostable polymerase. The PCR products
had been sequenced and compared with the data on PubMed. Then the amplified
fragments were purified and diluted to about 2 mg/L for microinjection. 346
fertilized oocytes were microinjected and 294 surviving injected oocytes were
implanted into 11 pseudopregnant mice. Among 34 newborns, four mice are PCR
positive and named 4, 5, 7 and 10 respectively (Fig. 2). All of them were
verified by Southern blot. The microinjected DNA fragments were integrated into
mouse genome and verified by the proof that about 50% of the offspring were PCR
positive. Quantitative Southern blot analysis was performed using equal amounts
of BglII-digested DNA, prepared from
tail biopsies of 4-week-old F1 transgenic mice belonging to each pedigree
(Fig.3). The copy numbers of integrated transgene are 15, 6, 2, and 4 for line
4, 5, 7 and 10, respectively.
Fig. 1 The constructed plasmid
Containing three elements: the human
scavenger enhancer (hESR), the metallothionein promoter
(PMT) and the mouse mSR cDNA sequence.
Fig. 2 PCR analysis of the MSR transgenic mice
4, 5, 7, 10 were
positive. The other negative results not shown. 1–10, genomic DNA from
different new born mice lines; C, negative control; P, microinjected DNA; H,
human genomic DNA. The pair of primers used here are hSR-enhancer.
Fig. 3 Southern blot
analysis of DNA from SR-1 transgenic mice progeny
The
arrow shows the correct position. The copy numbers of integrated transgene were
15, 6, 2 and 4, respectively. P, plasmid DNA; C, control mouse genomic DNA; 4,
5, 7 and 10, genomic DNA of different transgenic mice lines. All DNA were digested
with BglII.
2.2
Northern blot and RT-PCR
The previous studies have demonstrated
that the combination of the mSR promoter and a 400-bp upstream enhancer
is sufficient to confer macrophage-specific expression in mice[19].
In the present study we replaced the SR promoter by the metallothionein
promoter in order to induce the gene expression. The line 7 transgenic mouse
was used for the continued experiments. After one week’s induction, total RNA
was extracted from different tissues including liver, spleen, brain, intestine
and kidney. The RNAs of various tissues were examined by Northern blotting for
expression of the mSR using the cDNA probe (Fig. 4). The highest
expression was observed in spleen. There was faint signal in the liver and no
expression detected in brain, intestine and kidney. The result is somewhat
different from previous data in which several organs, such as kidney, showed
low expression of human mSR in the 180-kb YAC transgenic mice[14].
Comparing to the transgene we used, the 180-kb transgene is much longer and may
contain more regulatory elements so that the expression manner of the
transgenes is different between the two transgenic mice. Although the mouse MSR
is expressed predominantly in macrophage and would be detected in every tissues
by immunohistochemistry using specific anti-SR monoclonal antibodies[20],
only spleen was found to express detectable MSR in our study. The result
reflects that the percentage of macrophage of different tissues determines
whether the expression of the MSR can be detected at the whole organ level.
Fig. 4 Northern blot analysis of the
expression of mSR in multiple tissues
There is a strong and a weak signal in the lane S and L,
respectively. There is no signal in the line B, K and I. B, brain; S, spleen;
K, kidney; L, liver; I, ileum.
The metallothionein promoter has been
commonly used in the production of transgenic mice overexpressing secretory and
cell surface receptors[21,22]. The fused genes are presumed to
overexpress scavenger receptor in the macrophage-specific manner specifically.
In order to determine the efficiency of the zinc induction, the
semi-quantitation method, RT-PCR, were performed (Fig.5). The total RNAs were
extracted from peritoneal macrophages taken from zinc-induced transgenic mouse,
non-induced transgenic mouse, non-induced wild type and zinc-induced wild type
as control. The mouse G3PDH served as the internal control. The RT-PCR programs
were strictly operated in the same way except for different cDNA templates. The
quantity of the PCR products can be calculated by screening. The ratio of the
mSR to G3PDH indicates that there is no difference between non-induced and
zinc-induced wild type. It shows that only the ZnSO4 can’t affect
the level of the lipids in the plasma. At the same time the expression of mSR
in either transgenic mouse is moderately higher than that in the wild controls
(Table 1). There is no significantly difference between zinc-induced and
non-induced transgenic mouse. In the present study the effect of the
metallothionein promoter isn’t obvious as previous study[22].
Fig. 5 RT-PCR
analysis of the expression of the MSA
The upper bands show the
expression of mSR and the lower bands show the expression of mouse G3PDH
(the internal control). The result of the screening indicates that the
expression of mSR in either transgenic mouse is moderately higher than
that in the wild type controls. There is no significant difference between TZn
and T non (data not shown). TZn, zinc-induced transgenic
mouse; Tnon, non-induced transgenic mouse; W t, wild type controls.
Table 1. The serum triglyceride and cholesterol levels of
different mice
|
|
Triglyceride (g/L) |
Cholesterol(g/L) |
|
Wt ( |
0.88 + 0.27 |
1.09 + 0.23 |
|
Tnon ( |
0.91 + 0.20 |
0.64 + 0.20 |
|
TZn ( |
1.03 + 0.21 |
0.54 + 0.09 |
The serum triglyceride of
transgenic mouse is higher than wild type controls and it increases higher
after zinc induction. But the levels of serum cholesterol of transgenic mice
were significantly lower than the wild type controls (P<0.005). There is no sexual difference (data not shown). W
t, wild type controls; Tnon, transgenic and non-induced; TZn,
transgenic and zinc-induced.
2.3 Lipid profiles of mice expressing the
mouse mSR
After one day fasting, the blood samples were taken from the mice’s
tails. The serum triglyceride and total cholesterol were detected (Table 1).
There was no difference between male and female (data not shown). The results
indicated that the serum triglyceride of transgenic mouse is higher than wild
type controls and it increases much higher after zinc induction. On the other
hand, the total cholesterol of induced transgenic mouse decreased to half of
that of the wild control. Furthermore, the serum cholesterol of non-induced
transgenic mouse is also significantly different from that of the controls (P<0.005). These observations support
the idea that the excessive expression of the mSR can really affect the
serum lipids levels, especially the cholesterol, and its effect is completely
opposite for triglyceride and cholesterol, respectively. Interestingly, a
little increase of the expression of the gene can affect the serum lipids so
obviously. Further studies are needed to investigate the mechanism. In the
present study the mSR seems to decrease the concentration of serum cholesterol
and may be anti-atherogeneic, which is in accordance with previous data that
the overexpression of mSR in bone marrow-derived cells induced a
significant reduction in serum cholesterol level[23]. The possible
explanation is that SR-A can reduce the serum cholesterol levels by increasing
the clearance of lipoproteins. We suggest the mSR is multifunctional in the
atherogenesis. It can promote the pathogenesis of the disease by endocytosis of
modified lipoproteins, and it may also be anti-atherogeneic through other
undefined mechanisms, maybe the clearance of the natural lipoprotein. Further
research is needed to verifying that the overexprssion of SR-A can
antiatherogenesis on the high-cholesterol diet.
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