http://www.abbs.info e-mail:[email protected] ISSN 0582-9879                                          ACTA BIOCHIMICA et BIOPHYSICA SINICA 2003, 35(2): 117-121                                      CN 31-1300/Q

　

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, hE_SR 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 hE_SR was amplified by PCR using the human genomic DNA as templete and inserted into the KpnI site of the pGL3-P_MT-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 μC_i [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 hE_SR and metallothionein promoter (P_MT) 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 (hE_SR), the metallothionein promoter (P_MT) 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 T_Zn and T _non (data not shown). T_Zn, zinc-induced transgenic mouse; T_non, non-induced transgenic mouse;  W _t, wild type controls.

Table 1. The serum triglyceride and cholesterol levels of different mice

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; T_non, transgenic and non-induced; T_Zn, 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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