Liver LDL Receptor mRNA Expression Is Decreased in Human ApoB/CETP Double Transgenic Mice and Is Regulated by Diet as Well as the Cytokine Oncostatin M
Abstract We have investigated liver LDL receptor mRNA expression in nontransgenic, human cholesteryl ester transfer protein (CETP) transgenic, and human apolipoprotein (Apo) B/CETP double transgenic mice fed a normal chow diet and a high fat, high cholesterol diet (HFHC). Three weeks of HFHC feeding increased total serum cholesterol 1.5-fold in the nontransgenic, 3.1-fold in the CETP transgenic, and 3.4-fold in the ApoB/CETP double transgenic mice. To examine the liver LDL receptor mRNA expression among the different groups of mice fed the normal diet or fed the HFHC diet, we developed a quantitative reverse-transcribed polymerase chain reaction assay in which the LDL receptor mRNA level was normalized with the β-actin mRNA. The results show that on the normal chow diet, the LDL receptor mRNA expression levels were lower in the ApoB/CETP mice than in the nontransgenic mice and the human CETP transgenic mice. Liver LDL receptor gene expression was lower in all groups of mice fed the HFHC diet, with the lowest level of expression in the ApoB/CETP mice. Similar results were obtained by Northern blot analysis. In addition, we have previously shown that the cytokine oncostatin M (OM) increases LDL receptor gene expression in HepG2 cells. In this study, we used the ApoB/CETP mice as the model system to examine the in vivo activity of OM on liver LDL receptor gene expression. Our data show that OM increased the level of liver LDL receptor mRNA up to 80% to 90% when the animals were fed the HFHC diet. The results from these studies demonstrate that the expression of the liver LDL receptor in the ApoB/CETP mice is suppressed compared with nontransgenic mice and that the expression of the hepatic LDL receptor gene in these mice is subjected to the normal cholesterol feedback regulation. In addition, LDL receptor gene expression in these mice is also inducible by a positive regulator.
- Received February 10, 1997.
- Accepted July 15, 1997.
High levels of serum cholesterol are a major risk factor for coronary heart disease. Evidence accumulated through epidemiological studies, pathological investigations, clinical trials, and animal research has indicated that LDL, the major cholesterol-carrying lipoprotein in the serum, contributes significantly to this risk of coronary heart disease. In humans, at least 75% of circulating LDL is removed via LDL receptor mediated uptake in the liver. Therefore, hepatic LDL receptor plays a central role in cholesterol homeostasis and the development of coronary heart disease.
The importance of the liver LDL receptor in controlling serum cholesterol level has been demonstrated in animal models. In transgenic mice that overexpress liver LDL receptor, total serum cholesterol levels are less than 50% of those in their normal siblings, and plasma cholesterol levels are not significantly increased with HFHC feeding.1 In contrast, the mice that lack functional LDL receptor as a result of gene knockout accumulate significant amounts of LDL cholesterol in their plasma.2 These mice are also highly susceptible to the formation of atherosclerotic vascular lesions when fed with a cholesterol-rich diet.
The expression of LDL receptor is regulated predominantly through the cholesterol negative feedback pathway.3 Uptake of plasma LDL cholesterol results in an elevated intracellular cholesterol concentration, which suppresses the transcription of LDL receptor in a well-characterized example of end-product feedback repression. However, for studies on LDL uptake and LDL receptor regulation, normal laboratory mice are not optimal models because the LDL levels in these mice are extremely low. In the past decade, by using transgenic technology, several genetically engineered mouse models have been developed to study lipoprotein metabolism as well as atherosclerosis. Among these examples are mice that express human lipoprotein Apo B and mice that express human CETP. The ApoB transgenic mice have plasma ApoB-100 levels similar to those of normolipidemic humans. Human ApoB-100 was found in the LDL fraction in these mice, and LDL cholesterol levels were greatly increased compared with nontransgenic littermates.4 5 Human CETP is a 74-kD plasma glycoprotein that mediates the transfer of cholesteryl ester from HDL to VLDL and LDL particles.6 Although CETP is highly expressed in humans, it is not normally expressed in mice. Expressing human CETP in transgenic mice results in the redistribution of serum cholesterol from HDL to VLDL and LDL.7 8 Both the CETP and the ApoB single transgenic mice were shown to be susceptible to the development of atherosclerotic lesions on an HFHC diet.9 10
More recently, transgenic mice expressing both human ApoB and CETP were developed.11 When fed a normal diet, these mice have a lipoprotein-cholesterol distribution similar to that of normolipidemic humans, which makes them very useful for studying lipid metabolism and for examining a variety of pharmacological agents for their effects on lipoprotein cholesterol metabolism. It has been shown previously that the liver LDL receptor mRNA expression of a different line of CETP transgenic mice was lower than that of nontransgenic littermates.12 However, no information is available regarding the expression and regulation of the liver LDL receptor in these ApoB/CETP double transgenic mice.
It was previously reported that recognition of LDL receptor by its ligand apparently is species specific, namely that human LDL binds to the mouse LDL receptor with relatively low affinity.13 Since hepatic LDL receptor has a crucial effect on serum cholesterol level, we were interested in determining whether the high level of serum LDL cholesterol due to the expression of the human ApoB gene would affect the expression of the liver LDL receptor in these mice. Furthermore, we were also interested in determining whether LDL receptor expression in these mice could be regulated by dietary or pharmacological agents that have been shown to regulate LDL receptor in other animal models or in cell culture. Therefore, we have investigated the liver LDL receptor mRNA levels in nontransgenic, CETP transgenic, and ApoB/CETP double transgenic mice fed a normal chow diet and a high fat diet containing 1.25% cholesterol (HFHC). In these studies, two different approaches, RT-PCR and Northern blot analysis were used.
In addition to examining the effect of high LDL cholesterol levels on liver LDL receptor expression in these mice, we also examined the effect of the cytokine OM on LDL receptor expression in these mice. OM is a 28-kD glycoprotein predominantly produced by activated mononuclear leukocytes.14 15 16 OM was shown to upregulate cell surface LDL receptor density and LDL uptake in a human liver derived cell line HepG2.17 Research conducted in our laboratory has demonstrated that OM increases LDL receptor gene transcription in HepG2 cells cultured in medium containing high concentrations of cholesterol and 25-hydroxycholesterol.18 19 To investigate whether OM has the ability to increase LDL receptor transcription under conditions in which the LDL receptor is normally suppressed in vivo, we used the ApoB/CETP double transgenic mice as our model system and the quantitative RT-PCR method as a tool to examine the effect of OM on the mRNA levels of liver LDL receptor in individual mice.
Mice and Diets
The human CETP, ApoB/CETP double transgenic mice, and their nontransgenic littermates (C57BL/6J×SJL) have been characterized and described previously.11 All of the mice were housed in metabolic cages and given free access to food and water. The normal chow diet was obtained from PMI Feeds Inc, and the high fat diet containing 1.25% cholesterol (HFHC) was obtained from ICN Biomedicals. For OM administration, ApoB/CETP mice fed the high fat diet for 3 weeks were injected subcutaneously with human recombinant OM purified from Chinese hamster ovary cells20 at a dose of 10 μg/d with two injections (5 μg of OM in 100 μL of 1 mg/mL BSA/PBS) at 7 am and 3 pm for 7 days.
Serum Isolation and Cholesterol Determination
Mice were bled through the retroorbital plexus in the morning. For serum isolation, the blood was incubated at room temperature for 20 to 30 minutes before centrifugation at 14 000 rpm for 10 minutes. Standard enzymatic methods were used to determine total serum cholesterol (Sigma Chemical Co). Each serum sample was assayed in duplicate.
Fast Protein Liquid Chromatography Size Exclusion Analysis
Fifty microliters of serum pooled from eight mice was chromatographed on a Superose 6 column (Pharmacia Fine Chemicals). The column was equilibrated with 10 mmol/L Tris-HCl, pH 7.4, 0.15 mol/L NaCl, 0.01% (wt/vol) EDTA, 0.02% (wt/vol) NaN3 and run at a flow rate of 0.5 mL/min. Fractions of 0.5 mL were collected and 0.1 mL per fraction was used for cholesterol assay.
Total RNA Isolation and Northern Blot Analysis
Mice were sacrificed by cervical dislocation and the liver was surgically removed. On removal, 5 mL of RNA isolation reagent (ULTRASPEC RNA, Biotecx Laboratories) was immediately added to one lobe of the liver and the tissue was homogenized by a Brinkmann Polytron. One milliliter of the homogenization solution was used to proceed with the isolation of total RNA according to the vendor’s protocol. Fifteen micrograms of total RNA from each sample was separated on a 1% formaldehyde agarose gel. RNA was transferred to a Hybond N membrane by capillary action and was covalently linked to the membrane by UV cross-linking. Prehybridization and hybridization steps were performed under the conditions previously described.21 The blot was hybridized at 60°C to a 275-bp 32P-labeled murine LDL receptor probe generated by RT-PCR from the total RNA of mouse liver. The probe was labeled using 40 μCi [α-32P]dCTP with random-primed DNA labeling kit (Boehringer Mannheim Corp). The membrane was washed three times at ambient temperature with 2× SSC, 0.1% SDS and twice at 60°C with 0.1× SSC, 0.1% SDS. The membrane was then dried and exposed to X-OMAT scientific imaging film (Kodak) for 1 week at −80°C. Then the membranes were reprobed with a human GADPH probe to normalize the differences in the amounts of RNA from each sample. The autoradiographs were scanned by a laser densitometer (Personal Densitometer SI, Molecular Dynamics) and the integrated intensity of each band was analyzed with the program ImageQuaNT, version 4.1.
Optimal oligonucleotide primer pairs for PCR were selected with the aid of the program DNASIS (Hitachi Software Engineering America, Ltd). The primer sequence (5′ to 3′) for the murine LDL receptor gene coding (+) strand was GTG TGA AGA TAT TGA CGA GTG, and the noncoding (−) strand was AGT AGA TTC TAT TGT TGG TCA. The primer sequence for the murine β-actin gene coding (+) strand was ATG GTG GGA ATG GGT CAG AAG, and the noncoding (−) strand was CAC GCA GCT CAT TGT AGA AGG. The lengths of RT-PCR products were 275 bp for LDL receptor and 157 bp for β-actin.
The RT reaction was performed with a kit from Stratagene, using the following procedure: 19.3 μL of RNA (200 ng) was mixed with 1.5 μL of 9 base random primer (0.1 μg/μL) and incubated at 65°C for 5 minutes. After the reaction mixture cooled down slowly to room temperature, 2.5 μL of 10× first strand buffer, 0.5 μL of RNAse blocker, 1 μL of 100 mmol/L dNTPs, and 0.2 μL of MMLV-reverse transcriptase (50 U/μL) were added. The mixture was incubated at 37°C for 1 hour and then 90°C for 5 minutes.
The PCR reaction was performed with the PCR reagents obtained from Perkin-Elmer. For detection of the LDL receptor gene, the reaction mixture contained 4 μL of cDNA (RT reaction mixture), 8 μL of 10× Taq DNA polymerase buffer, 0.64 μL of 100 mmol/L dNTP, 0.6 μL of coding (+) strand primer (0.1 μg/μL ), 0.6 μL of noncoding (−) strand primer (0.1 μg/μL ), 66 μL of RNAse-free H2O, and 0.4 μL of Taq DNA polymerase (5 U/μL). The PCR reaction was performed on a temperature cycler (TwinBlock System, ERICOMP) using the following conditions: The samples were denatured at 92°C for 5 minutes, followed by 34 cycles of 91°C for 1 minute, 55°C for 1 minute, 72°C for 2 minutes. Finally, the reactions were incubated at 72°C for 10 minutes to extend any incomplete single strands. For detection of β-actin gene, the reaction mixture contained 2 μL of cDNA, 8 μL of 10× Taq DNA polymerase buffer, 0.64 μL of 100 mmol/L dNTP, 1.2 μL of upper-strand primer (0.1 μg/μL ), 1.2 μL of lower-strand primer (0.1 μg/μL ), 67 μL of RNAse-free H2O, and 0.4 μL of Taq DNA polymerase (5 U/μL). The PCR reaction was conducted as described for the LDL receptor gene, except that 25 cycles were used to amplify the product.
After the PCR reaction, 50 μL of the reaction mix was transferred to a fresh tube and mixed with 10 μL of 6× DNA loading buffer. Twenty-five microliters per sample were then electrophoresed on a 2% SeaKem agarose gel (FMC BioProducts). The gels were stained with ethidium bromide at a concentration of 0.4 μg/mL for 30 minutes and photographed with Kodak TMA ×100 negative film (4×5 in) with a Polaroid MP-4 camera under the UV transilluminator. The photographic negatives were scanned and analyzed as described in the Northern blot analysis. The relative signal of LDL receptor mRNA was normalized to the signal of β-actin mRNA and was expressed as the ratio of LDL receptor/β-actin.
The above-described RT-PCR reactions for detection of LDL receptor and β-actin messengers were determined on the basis of a series of experiments to test the reaction conditions, such as the amount of RNA linearity, cycle linearity, primer concentrations, and generation of standard RT-PCR curves for LDL receptor and β-actin. The final RT-PCR conditions were shown to be in the linear range of the amplification for both genes.
Comparisons of experimental data were analyzed by a two-tailed Student’s t test and the Newman-Keuls multiple comparison test. A value of P<.05 was considered to indicate a statistically significant difference.
The effects of an HFHC diet on total serum cholesterol were examined in male nontransgenic (C57BL/6J×SJL; non-Tg), CETP transgenic (CETP), and ApoB/CETP double transgenic (ApoB/CETP) mice. The mice were fed either normal laboratory chow or a high fat diet containing 1.25% cholesterol (HFHC) for 3 weeks. As shown in Fig 1A⇓, when fed a normal chow diet, the total serum cholesterol levels were similar between the non-Tg and the ApoB/CETP transgenic mice and were slightly higher than those of the CETP mice. After 3 weeks on the HFHC diet, total serum cholesterol levels were increased in all the groups, but the extents of the increases were significantly different when comparing the nontransgenic and transgenic animals. The total serum cholesterol was increased 1.5-fold in the non-Tg mice (73.5 mg/dL to 113.8 mg/dL, P=.023), 3.1-fold in the CETP mice (50.9 mg/dL to 160.4 mg/dL, P=.025), and approximately 3.4-fold in the ApoB/CETP mice (73.3 mg/dL to 253.6 mg/dL, P=.010). A similar finding was obtained in female mice (Fig 1B⇓). However, the baseline serum cholesterol (chow) of the ApoB/CETP mice fed the normal chow diet was significantly higher than that of non-TG and CETP mice (P<.001). The differences of the total serum cholesterol levels between the male ApoB/CETP mice and the female ApoB/CETP mice both were statistically significant under the normal chow (P<.05) and the HFHC diet (P<.001).
To examine the LDL cholesterol levels and the lipoprotein profiles of the ApoB/CETP mice fed the HFHC diet and the normal chow diet, fast protein liquid chromatography analysis of the pooled serum samples from these mice fed the normal chow diet or the HFHC diet was performed. The results were compared with the normal mice fed the normal chow diet and the HFHC diet. When fed the normal chow diet, only the serum from ApoB/CETP mice contained a high proportion of LDL cholesterol, with approximately a 2-to-1 ratio of LDL cholesterol to HDL cholesterol (Fig 2A⇓). On the HFHC diet, total serum cholesterol levels increased in both the Apo B/CETP mice and the non-Tg mice, and the majority of increase was observed in the VLDL fraction (Fig 2B⇓). However, after a longer period of HFHC feeding (6 months), LDL cholesterol levels were also elevated in the ApoB/CETP mice (data not shown).
To determine whether the high circulating levels of LDL cholesterol would affect the liver LDL receptor expression in the ApoB/CETP mice fed either the normal chow diet or the HFHC diet, non-Tg mice (n=3 per diet), CETP transgenic mice (n=5 per diet), and ApoB/CETP double transgenic mice (n=3 per diet) were placed on either of the above diets. Male mice were sacrificed after 3 weeks, the livers were removed, and total RNAs were isolated. To determine the LDL receptor mRNA levels, we developed an assay that used RT-PCR to quantitate LDL receptor mRNA from the mouse liver. In this study, a housekeeping gene, β-actin, was coamplified with LDL receptor from each RNA sample to normalize the amount of template RNA. Subsequently, the PCR products were separated on a 2% agarose gel. As shown in Fig 3A⇓, RT-PCR generated a 275-bp DNA fragment from LDL receptor mRNA and a 157-bp DNA fragment from β-actin mRNA. Relative LDL receptor signals normalized to β-actin were determined by laser densitometric analysis of the photographic negatives from agarose gels stained with ethidium bromide. The quantitative data are presented in Fig 3B⇓. The results of the RT-PCR analysis indicated that liver LDL receptor mRNA expression was decreased in all three groups when fed the HFHC diet. The LDL receptor mRNA levels were 52%, 30%, and 58% lower in non-TG mice, CETP transgenic, and ApoB/CETP double transgenic mice, respectively, when fed the HFHC diet. Furthermore, among the three groups, ApoB/CETP mice expressed lower levels of liver LDL receptor mRNA compared with the non-Tg and CETP mice under both diets. In comparison, the expression of the housekeeping gene β-actin was not different among the groups and was not affected by the diet. A similar result was obtained by Northern blot analysis (data not shown).
Previously, we have shown that the cytokine OM increased LDL receptor mRNA level and LDL receptor promoter activity in the human liver–derived cell line HepG2.17 18 More interestingly, we found that the OM activity on LDL receptor gene transcription was not diminished in culture medium containing a high concentration of sterol. We were interested in examining the effect of OM on liver LDL receptor in an animal model. Since the lipoprotein profile of the ApoB/CETP mice resembles that of the normolipidemic human and our data show that the high cholesterol feeding increased total serum cholesterol and suppressed liver LDL receptor mRNA levels, it is likely that the liver LDL receptor in these mice is subjected to normal cholesterol feedback regulation. Therefore, we decided to use ApoB/CETP mice to investigate the in vivo activity of OM on liver LDL receptor mRNA expression.
Male ApoB/CETP mice (n=8) were fed the HFHC diet for 3 weeks. Human recombinant OM was then administered subcutaneously at a daily dose of 10 μg per mouse (n=4) with two injections (7 am and 3 pm) for 7 days. The control mice (n=4) received the same treatment with the OM dilution buffer (100 μL of 1 mg/mL BSA in PBS). Liver total RNA was recovered from OM-treated and control mice. Specific detection of mouse liver LDL receptor mRNA was accomplished by using the RT-PCR method as described in Fig 3⇑. As shown in Fig 4⇓, OM increased the level of liver LDL receptor mRNA up to 80% and 87% in two independent RT-PCR reactions. Because the expression of LDL receptor mRNA was further suppressed by the HFHC feeding in these ApoB/CETP mice that already express the lowest level of hepatic LDL receptor mRNA compared with nontransgenic mice and also in the CETP transgenic mice, we were unable to accurately measure the LDL receptor mRNA levels in control and OM-treated mice by Northern blot analysis due to the low sensitivity of the assay.
Previous studies have shown that the human ApoB/CETP double transgenic mice have a lipoprotein-cholesterol profile similar to that of normolipidemic humans. When fed a normal chow diet, these mice express human ApoB-100 at the level of a normolipidemic human and have significantly higher levels of LDL cholesterol.11 In the current study, we have investigated the expression and regulation of the hepatic LDL receptor mRNA in these ApoB/CETP transgenic mice. We have used two independent approaches, RT-PCR analysis and Northern blot analysis, to perform these experiments.
Initially, we compared the cholesterol levels of nontransgenic, CETP transgenic mice, and ApoB/CETP transgenic mice fed a normal chow diet or a HFHC diet. On a normal chow diet, the non-Tg and ApoB/CETP males have similar levels of total cholesterol. These levels were slightly higher than those of the CETP males. Interestingly, the total serum cholesterol levels in the ApoB/CETP females were significantly higher than those observed in non-Tg and CETP mice (P<.001). To make a fair comparison of the differences of the total serum cholesterol levels between the male mice and the female mice of the different groups, we used the Newman-Keuls multiple comparison test, and the results show that only the male ApoB/CETP mice and the female ApoB/CETP mice were statistically significant different both under the normal chow (P<.05) and the HFHC diet (P<.001). Therefore, in light of these sex-related differences in the cholesterol levels in these mice, our studies comparing LDL receptor expression in these different groups of mice were performed with sex-matched animals.
The initial studies characterizing the ApoB/CETP mice did not examine the effects of the HFHC diet on total serum cholesterol. It is important to know how the ApoB/CETP transgenic mice respond to the high cholesterol–containing diet compared with the normal mice. Therefore, in this study, the ApoB/CETP, CETP, and nontransgenic mice were fed the HFHC diet for 3 weeks, and the total serum cholesterol levels were measured. The result showed that the total serum cholesterol levels were significantly increased in mice maintained on the HFHC diet. The most striking increases were observed in the ApoB/CETP double transgenic mice. Unlike nontransgenic and CETP transgenic mice, these mice had significant levels of cholesterol both in VLDL and in LDL (Fig 2B⇑). The increased cholesterol by HFHC feeding was mainly in the VLDL fraction in this study. However, in a separate study, the ApoB/CETP mice were fed the HFHC diet for a longer period of time (6 months), and the LDL cholesterol level was significantly increased. Based on these results, we feel confident that these mice are suitable for studying the effect of LDL cholesterol on LDL receptor expression, as well as studying cholesterol metabolism and possibly atherosclerosis.
The RT-PCR assay for mouse LDL receptor expression was developed as an important tool to quantify the expression of the LDL receptor gene. In this assay, coamplification of PCR products from LDL receptor mRNA with β-actin mRNA allowed normalization of the concentrations of total RNA template used in each reaction. Since the quantitative application of this method is contingent on analysis of the PCR products during the linear phase of the amplification, a series of preliminary experiments was conducted to optimize the reactions and determine the linear range. The results of the RT-PCR analysis were similar to those obtained from the Northern analysis, indicating that the HFHC diet significantly decreased LDL receptor mRNA level (P=.01) in the ApoB/CETP mice. The HFHC diet also lowered the liver LDL receptor mRNA in the nontransgenic mice (P=.023) and in the CETP mice (P=.025). In addition, these studies demonstrated that liver LDL receptor mRNA expression in ApoB/CETP mice was decreased compared with that of nontransgenic littermates, although this did not quite reach statistical significance when the animals were fed a normal chow diet (P=.07). However, when the animals were fed the HFHC diet, the difference was significant (P=.05). When fed a normal chow diet, the total cholesterol levels were similar for both the ApoB/CETP mice and the nontransgenic mice. The reduction of LDL receptor expression in this case is likely to be related to the relative high levels of LDL cholesterol in the serum of these mice. It has been shown that in an in vitro assay, the human ApoB molecule does not interact with the murine LDL receptor expressed in a fibroblast cell line as efficiently as the murine ApoB molecule. Our current results suggest that the human ApoB protein can interact sufficiently with the hepatic LDL receptor to increase intracellular levels of cholesterol and downregulate the expression of the LDL receptor in vivo. This difference may be related to the different assay systems, namely in vivo versus in vitro, and different cell types. Alternatively, intracellular cholesterol levels are increased through a mechanism independent of the LDL receptor. For example, the murine LDL in the transgenic mice contain ApoE that could mediate the interaction of VLDL and LDL with the murine LDL receptor. The expression of the hepatic LDL receptor mRNA in all of the groups of mice was decreased when the mice were fed the HFHC diet. This is not surprising, as levels of VLDL cholesterol were increased in all three groups when fed the HFHC diet. Under these conditions, however, the ApoB/CETP mice still possessed the lowest levels of hepatic LDL receptor mRNA, suggesting that in this system, LDL cholesterol is capable of further lowering the LDL receptor expression levels.
We have shown previously that OM increases LDL receptor gene expression in HepG2 cells. OM increases the LDL receptor mRNA and LDL receptor promoter activity up to threefold in HepG2 cells at 1 to 3 nmol/L concentration.19 In this study, we showed that administration of human OM (10 μg per mouse) in ApoB/CETP transgenic mice resulted in a significant increase of liver LDL receptor mRNA (87% and 80% over control in two independent RT-PCR assays on mice fed with the high cholesterol diet. Future studies will focus on identifying the optimal dose and duration of treatment for maximizing the effect of OM on LDL receptor mRNA expression and serum cholesterol levels.
In summary, this study has shown that in the ApoB/CETP double transgenic mice, the hepatic LDL receptor can be negatively regulated by high serum LDL cholesterol and upregulated by the cytokine OM in a manner similar to the cholesterol-LDL receptor interactions described in the human liver cells in vitro.17 18 19 In addition, this study indicates that LDL cholesterol levels in these ApoB/CETP double transgenic mice can affect LDL receptor expression. These results were determined by two independent approaches, quantitative RT-PCR and Northern blot analysis. Our demonstration that LDL receptor gene expression can be activated or suppressed suggests that these mice may be useful as a model for developing new cholesterol-lowering drugs.
Selected Abbreviations and Acronyms
|CETP||=||cholesteryl ester transfer protein|
|HFHC||=||high fat, high cholesterol (dict)|
|RT-PCR||=||reverse-transcribed polymerase chain reaction|
This study was supported by a grant from the American Heart Association (No. 9507833S) and by the Department of Veterans Affairs (Office of Research and Development, Medical Research Service). We would like to thank Urmil Saini and Kristin Forcier for their excellent technical assistance and Dr Mark E. Swanson at Chrysalis DNX Transgenic Sciences and Dr Michael R. Briggs at Ligand Pharmaceuticals for stimulating discussions.
Yoked M, Hammer RE, Ishibashi S, Brown MS, Goldstein JL. Diet-induced hypercholesterolemia in mice: prevention by overexpression of LDL receptors. Science. 1990;250:1273.
Ishibashi S, Brown MS, Goldstein JL, Gerard RD, Hammer RE, Herz J. Hypercholesterolemia in LDL receptor knockout mice and its reversal by adenovirus-mediated gene delivery. J Clin Invest. 1993;92:883.
Linton MF, Farese RV Jr, Chiesa G, Grass DS, Chin P, Hammer RE, Hobbs HH, Young SG. Transgenic mice expressing high plasma concentrations of human apolipoprotein B100 and lipoprotein(a). J Clin Invest. 1993;92:3029.
Xiong W, Zsigmond E, Gotto AM Jr, Lei KY, Chan L. Location of a low density lipoprotein-targeting domain of human apolipoprotein B-100 by expressing a minigene construct in transgenic mice. J Biol Chem. 1991;266:20893.
Agellon LB, Walsh A, Hayek T, Moulin P, Jiang XC, Shelanski SA, Breslow JL, Tall AR. Reduced high density lipoprotein cholesterol in human cholesterol ester transfer protein transgenic mice. J Biol Chem. 1991;266:10796.
Marotti KR, Castle CK, Murray RW, Rehberg EF, Polites HG, Melchior GW. The role of cholesteryl ester transfer protein in primate apolipoprotein A-I metabolism: insights from studies with transgenic mice. Arterioscler Thromb. 1992;12:736.
Purcell-Huynh DA, Farese RV Jr, Johnson DF, Flynn LM, Pierotti V, Newland DL, Linton MF, Sanan DA, Young SG. Transgenic mice expressing high levels of human apolipoprotein B develop severe atherosclerotic lesions in response to a high-fat diet. J Clin Invest. 1995;95:2246.
Grass DS, Saini U, Felkner RH, Wallace RE, Lago WJP, Young SG, Swanson ME. Transgenic mice expressing both human apolipoprotein B and human CETP have a lipoprotein cholesterol distribution similar to that of normolipidemic humans. J Lipid Res. 1995;36:1082.
Jiang XC, Masucci ML, Mar J, Lin M, Walsh A, Breslow JL, Tall A. Down-regulation of mRNA for the low density lipoprotein receptor in transgenic mice containing the gene for human cholesteryl ester transfer protein: mechanism to explain accumulation of lipoprotein B particles. J Biol Chem. 1993;268:27406.
Corsini A, Mazzotti M, Villa A, Maggi FM, Bernini F, Romane L, Romano C, Fumagalli R, Catapano AL. Ability of the LDL receptor from several animal species to recognize the human apo B binding domain: studies with LDL from familial defective apo B-100. Atherosclerosis. 1992;93:95.
Zarling JM, Shoyab M, Marquardt H, Hanson MB, Lioubin MN, Todaro GJ, Oncostatin M: a growth regulator produced by differentiated lymphoma cells. Proc Natl Acad Sci U S A. 1986;83:9739.
Brown TJ, Lioubin MN, Marquardt H. Purification and characterization of cytostatic lymphokines produced by activated human T-lymphocytes: synergistic antiproliferative activity of transforming growth factor β1, interferon gamma, and oncostatin M for human melanoma cells. J Immunol. 1987;139:2977.
Grove RI, Mazzucco C, Allegretto N, Kiener PA, Spitalny G, Radka SF, Shoyab M, Antonaccio M, Warr GA. Macrophage-derived factors increase low-density lipoprotein uptake and receptor number in cultured human liver cells. J Lipid Res. 1991;32:1889.
Grove RI, Mazzucco CE, Radka SF, Shoyab M, Kiener PA. Oncostatin M upregulates LDL receptor in HepG2 cells by a novel mechanism. J Biol Chem. 1991;266:18194.
Liu J, Grove RI, Vestal RE. Oncostatin M activates the LDL receptor gene transcription in sterol-repressed liver cells. Cell Growth & Differentiation. 1994;5:1333.
Liu J, Streiff R, Zhang YL, Robert E, Vestal Spence, MJ, Briggs M. Novel mechanism of transcriptional activation of hepatic LDL receptor by oncostatin M. J Lipid Res. In press.
Malik N, Graves D, Shoyab M, Purchio AF. Amplification and expression of heterologous oncostatin M in Chinese hamster ovary cells: DNA. Cell Biol. 1992;11:453.