Quantitative Trait Locus Mapping of Genes That Regulate Phospholipid Transfer Activity in SM/J and NZB/BlNJ Inbred Mice
Objective Phospholipid transfer protein (PLTP), an important protein in the transfer of phospholipids between lipoprotein particles and in the remodeling of HDL, is regulated at both the transcriptional and the protein level. We performed quantitative trait locus (QTL) analysis to identify genomic loci regulating PLTP activity in mice.
Methods and Results Plasma PLTP activity was measured in 217 male F2 progeny from a SM/J × NZB/B1NJ intercross. Two QTL for plasma PLTP activity in mice fed chow (Pltpq1 and Pltpq2) were found on chromosomes 3 (34 cM, logarithm of odds [LOD] 3.5) and 10 (66 cM, LOD 4.1); two additional QTL in mice fed atherogenic diet (Pltpq3 and Pltpq4) were found on chromosomes 9 (56 cM, LOD 4.5) and 15 (34 cM, LOD 5.0); and one QTL (Pltiq1) for the inducibility of PLTP activity was found on chromosome 4 (70 cM, LOD 3.7). Several candidate genes for these 5 QTL were tested by sequence comparison and expression studies.
Conclusions We identified five significant loci involved in PLTP activity in the mouse and provided supporting evidence for the candidacy of Nr1h4 and Apof as the genes underlying Pltpq2.
Plasma phospholipid transfer protein (PLTP) is responsible for the transfer of phospholipids from very low-density lipoproteins (VLDL) to high-density lipoproteins (HDL)1 and remodels HDL into larger and smaller particles, generating prebeta HDL.2 PLTP is believed to be an important factor in the development of cardiovascular disease, but its precise role is not understood. Recently published reports assigned both atherogenic and antiatherogenic properties to PLTP. For example, mice with a hyperlipidemic background show a decrease in atherosclerosis susceptibility when PLTP is knocked out.3 On the other hand, PLTP enhances the removal of cellular cholesterol and phospholipids4 and PLTP overexpression prevents accumulation of intracellular cholesterol in macrophages.5 Furthermore, PLTP mediates alpha-tocopherol transfer, which provides LDL with antioxidants and preserves the normal function of endothelial cells.6 More insight is needed to determine the relation between PLTP activity and the atherosclerotic process. Regulation of PLTP activity is an interesting target for future drug development and understanding the mechanism is a first step toward this goal.
Several transcription factor binding sites (sterol regulatory element-binding protein [SREBP], peroxisome proliferator–activated receptor [PPAR], and CCAT/enhancer-binding protein [C/EBP]) have been identified in the promoter region7 and PPAR alpha (PPARA), liver X receptor (LXR), and farnesoid X receptor (FXR) have been reported to be involved in PLTP transcription.8–10 But PLTP activity and mass in human plasma do not correlate, suggesting that there may be varying amounts of a catalytically inactive form of PLTP.11 Not much is known about the regulation at the protein level or the factors that are involved. The lipoprotein profile in plasma seems to influence the ratio between “active” and “inactive” PLTP,12 but the molecular basis and the functional differences between the two forms remains unclear.
To identify key loci that are involved in regulation of PLTP activity, we performed a quantitative trait locus (QTL) analysis on male F2 intercross mice between the SM/J and NZB/B1NJ inbred strains. These two strains differ significantly for PLTP activity,13 HDL levels,14 and atherosclerosis susceptibility.15 We report two QTL for PLTP activity in mice fed chow, two different QTL for mice fed atherogenic diet, and a QTL for inducibility using the log transformation of the ratio of the activities for both diets.
Mice and Diets
SM/J and NZB/BlNJ inbred mice were obtained from the Jackson Laboratory (Bar Harbor, Me). SM females were mated to NZB males to produce the F1 progeny; F1 mice were intercrossed to produce 217 male F2 progeny. Mice were housed in a climate-controlled facility with a 14-hour:10-hour light-dark cycle with free access to food and water throughout the experiment. After weaning, mice were maintained on a chow diet (Old Guilford 234A, Guilford, Conn) until 8 weeks of age and then fed an atherogenic diet for 6 weeks containing (w/w) 15% dairy fat, 50% sucrose, 20% casein, 0.5% cholic acid, 1.0% cholesterol, as well as cellulose, vitamins, and minerals. The source of chemicals and the diet have been described previously.16 All experiments were approved by the Jackson Laboratory’s Animal Care and Use Committee.
Phospholipid Transfer Protein Activity
At 0 and 6 weeks of diet consumption, mice were fasted for 4 hours, blood was collected by retro-orbital bleeding into EDTA-coated tubes, and plasma was separated by centrifugation at 1500 rpm for 5 minutes at 4°C. The plasma phospholipid transfer activity mediated by PLTP was determined by measuring the transfer of [14C]phosphatidylcholine from phospholipid liposomes to HDL3 as described previously.17 As phospholipid transfer activity in the mouse plasma of these two particular strains is considerably higher than in human plasma,13 only half the amount of plasma (0.5 μL) was assayed compared with human plasma to keep the assay in the linear range. We included in each assay a 50-μL aliquot of three different 1:50 diluted, human control plasma samples in quadruplicate. The amount of phospholipid transferred by plasma PLTP was calculated as percent of total radioactivity per assay tube transferred to HDL minus background transfer (tubes without PLTP source).
Genotyping and Real-Time Polymerase Chain Reaction
DNA was isolated and genotyping performed as described previously.14 Total RNA was extracted from livers of two groups of NZB and SM male mice: eight 8-week old mice fed a chow diet, and eight 12-week old mice fed the atherogenic diet for 4 weeks. Total RNA was obtained using the Trizol method (Invitrogen, Carlsbad, Calif) according to manufacturer’s recommendations and was converted to cDNA using the Omniscript RT kit (Qiagen, Valencia, Calif). Gene-specific primers (Table I, available online at http://atvb.ahajournals.org) were designed and tested for specificity by sequencing the polymerase chain reaction (PCR) product. To amplify and detect the target gene and the reference gene, we used the Quantitect SYBR green PCR kit (Qiagen) according to the manufacturer’s recommendations. Real-time PCR on cDNA was performed using an ABI Prism 7000 (Applied Biosystems, Foster City, Calif). Expression was calculated as the number of molecules per 1000 molecules of beta-2-microglobin (B2m) and the results are shown as the mean±SE.
Sequencing of Candidate Genes
To sequence the coding regions of the candidate genes Apoa1bp, Nr1h4, Apof, Scap, Ppara, and Nr0b2, primers were designed to span each of the exons for amplification using NZB/BlNJ, SM/J, 129S1/SvImJ, C57BL/6J, CAST/Ei, DBA2/J, C3H/HeJ, A/J, SJL/J, and SWR/J genomic DNA (Mouse DNA Resource, the Jackson Laboratory). Direct sequencing was performed on the PCR products using Big Dye Terminator Cycle Sequencing Chemistry and the ABI 3700 Sequence Detection System (Applied Biosystems). Results were analyzed using the Wisconsin Package (Accelrys Inc, San Diego, Calif).
We performed genome-wide scans for QTL using the method of Sen and Churchill;18 significance was determined by permutation testing for each trait.19 The software package Pseudomarker (release 9.1, Sen and Churchill, http://www.jax.org/staff/churchill/labsite) was used. Significant and suggestive QTL meet or exceed the 95% and 90% genome-wide thresholds, respectively. First, we performed one-dimensional genome scans on a single-QTL basis to detect QTL with main effects. Then, simultaneous genome scans for all pairs of markers were performed to detect epistatic interactions. Finally, all the detected main effect and interacting QTL were used to fit multiple regression models. The type III sum of squares of each marker or marker pair to the total sum of squares is the percentage of variance explained by each marker or marker pair. Other statistical analyses were done using Prism V3.02 (GraphPad Software, Inc). Between-group comparisons were analyzed by one-way ANOVA followed by Tukey HSD test to determine statistical significance. Correlation between measurements was tested using simple linear regression analysis.
PLTP Activity in Parental Strains, F1 and F2
The plasma PLTP activity in the parental strains and the F1 and F2 populations are summarized in Figure 1. PLTP activity in NZB mice was significantly higher than in SM mice. In F1 animals on chow, the F1 values were closer to NZB mice, whereas on atherogenic diet, F1 values more resembled SM mice. Mean PLTP activity in F2 progeny did not significantly differ from those of the F1 animals and the values were distributed normally around the mean on both diets (Figure 1).
Expression of Pltp in the liver was measured in the parental strains by quantifying Pltp mRNA using real-time PCR. NZB had significantly higher levels than SM (P<0.05) on both diets, consistent with the higher PLTP activity in plasma. No significant increase in Pltp expression occurred in mice fed the atherogenic diet (Figure 2A).
Identification of QTL for PLTP Activity
The genome-wide scan indicated significant QTL for PLTP activity in chow-fed mice on chromosomes 3 and 10 (Figure 3A) and were named Pltpq1 (LOD 3.5, Figure 4A) and Pltpq2 (LOD 4.1, Figure 4B), respectively (for Phospholipid transfer protein QTL). The Pltpq1 allele for high PLTP activity came from NZB and is additive. For Pltpq2, the heterozygous F2 animals had a significantly (P<0.05) higher PLTP activity than the animals that were homozygous for either SM or NZB. Interestingly, QTL for HDL cholesterol levels overlap with these two QTL in the same cross (Korstanje et al, in preparation).
Pltpq1 and Pltpq2 were not found when PLTP activity was measured after a 6-week atherogenic diet. Instead, two other significant QTL were found, Pltpq3 (LOD 4.5) on chromosome 9 and Pltpq4 (LOD 5.0) on chromosome 15 (Figures 3B, 4C, and 4⇑D). For Pltpq3, animals that were homozygous for the NZB allele had high PLTP activity, whereas heterozygous animals and animals homozygous for the SM allele had lower PLTP activity. For Pltpq4, animals that were homozygous for the SM allele had higher PLTP activity than animals that were homozygous for the NZB allele. Heterozygous animals were intermediate.
PLTP inducibility, defined as the logarithm of the ratio between the PLTP activities on atherogenic diet and chow diet, was affected by one significant QTL (LOD 3.7) on chromosome 4 (Figures 3C and 4⇑E); we named this Pltiq1 (for Phospholipid transfer protein inducibility QTL). The NZB allele for high inducibility was dominant over the SM allele. The QTL are summarized in Table 1.
The QTL on chromosome 10 was fitted with models comprising one, two, or three QTL, and a maximum LOD score calculated for each. Permutation testing was used to determine significance thresholds. Increases of 2.2, 1.6, and 1.4 or greater in the LOD score were the thresholds used to declare multiple QTL at the 95%, 90%, and 80% confidence levels, respectively. We observed an increase of 1.5 LOD (3.8 versus 5.2) for the one-QTL versus the two-QTL model, but an increase of only 0.5 LOD for the three-QTL model. Therefore, it is likely, but not conclusive, that two QTL on chromosome 10 contribute to PLTP activity. No evidence was found for multiple QTL for the other chromosomes. Tests for interaction between loci did not show significant interactions for any of the three phenotypes. The QTL found in this analysis explained 11.2%, 12.9%, and 7.9% of the variance of PLTP activity in chow-fed mice, atherogenic diet-fed mice, and the inducibility, respectively (multiple regression models for the QTL analyses [Table III, available online at http://atvb.ahajournals.org]).
Testing of Candidate Genes
We examined each QTL interval and selected several candidates for additional study (Table 1). Because either expression or functional differences in a gene can give rise to a QTL, we analyzed both the mRNA expression levels and the coding sequences of these candidate genes.
The candidate gene for Pltpq1, apolipoprotein AI binding protein (Apoa1bp), had no sequence differences in the coding region and no significant difference in expression in the liver between SM and NZB mice fed chow (Figure 2B). Therefore, this gene is unlikely to underlie the QTL.
NR1H4 (FXR), a candidate for Pltpq2, is a known transcription factor for Pltp.7 We identified 6 SNPs in the coding region of Nr1h4, three of them leading to conserved amino acid changes (Table II, available online at http://atvb.ahajournals.org). Nr1h4 showed a 10-fold increase in hepatic expression in NZB compared with SM mice fed either diet and a slight increase in SM fed the atherogenic diet compared with the chow diet (Figure 2C). Thus, the candidacy of Nr1h4 is supported by the expression difference between the two strains.
Another candidate gene for Pltpq2 is Apof, a lipid transfer inhibitor protein.20 We did not observe a difference in expression of Apof between NZB and SM, however, both strains showed a significant decrease in Apof expression on the atherogenic diet (Figure 2D). Two SNPs were found in the coding region of Apof. One difference, guanine in SM and cytosine in NZB at position 801, gives rise to a nonconservative amino acid difference at position 267 (on the basis of the first ATG as +1), a glycine to a histidine, in the structural part of the protein. Sequencing in other inbred strains shows that guanine is present in SM, C57BL/6J, DBA/2J, C3H/HeJ, MOLF/Ei, and CAST/Ei, and cytosine is present in NZB and 129SV/J. Thus, if Apof is the gene for Pltpq2, we suggest that the amino acid change may cause a functional difference in the protein.
The gene-encoding SREBP cleavage activating protein (Scap) is a candidate gene for Pltpq3, a QTL found only in atherogenic diet-fed mice. Expression analysis in the liver showed a two-fold difference in chow fed mice, but no difference in the atherogenic diet fed mice (Figure 2E). A sequence difference in the coding region of Scap leads to a conservative amino acid change. Thus, the lack of a difference in expression and the conservative amino acid change do not support the candidacy of Scap.
The gene encoding PPARA, another known transcription factor for Pltp,8 is a candidate gene for Pltpq4. Sequence differences were found in the 5′ and 3′ UTR of the gene, but not in the coding region, and there was no significant difference of Ppara expression in the liver between NZB and SM on the atherogenic diet. Interestingly, the expression level of Ppara in SM on chow was significantly higher than in NZB on chow (P<0.05) and higher than in SM on the atherogenic diet (Figure 2F).
Small heterodimeric partner (SHP) is a transcription factor involved in cholesterol metabolism and the gene (Nr0b2) is a candidate for Plti1. We found a significant difference between NZB and SM on the chow diet, but not on the atherogenic diet (Figure 2G). We also found a significant difference between SM on chow and on the atherogenic diet (P<0.05). Sequencing of the coding region revealed no differences between SM and NZB.
Urizar et al.10 found a significant increase in hepatic Pltp mRNA expression when feeding C57BL/6 male mice a diet containing 1% cholic acid. In the present study, SM and NZB male mice have no increased hepatic Pltp mRNA expression on a diet containing 0.5% cholic acid. One explanation why these results differ could be the difference in the amount of cholic acid in the diet. A better explanation is the reference gene used; we observed that hepatic beta-actin mRNA levels, which is the reference used by Urizar et al.,10 decreases because of a cholic acid diet (data not shown), causing an apparent increase in Pltp expression. This decrease of beta-actin made the gene, in our view, unfit as a reference. Therefore, we used a gene, B2m, whose expression levels are not affected by the diet change.
Because no QTL was found at the location of the Pltp gene (chromosome 2, 93 cM), we conclude that the difference in PLTP activity and the differential gene expression of hepatic Pltp between SM and NZB is because of differences in transacting elements that regulate PLTP activity and Pltp expression.
A locus associated with PLTP activity on the chow diet is Plptq1 on chromosome 3. A candidate gene, the ApoA-I binding protein (Apoa1bp), is located within the 95% confidence interval of this QTL. Because PLTP interacts with Apo A-I21, APOA1bp might be involved in this interaction. However, there is no evidence to support the candidacy of this gene; we found no sequence differences in the coding region and no difference in expression in the liver (Figure 2B).
Pltp can be regulated by the farnesoid X-activated receptor (FXR),10 which is encoded by Nr1h4, a candidate gene for Pltpq2. Higher expression levels of FXR lead to higher expression of PLTP, and NZB mice had a 10-fold higher expression of Nr1h4 in liver compared with SM mice (Figure 2C). We observed a small increase in expression in SM mice fed atherogenic diet; this might be enough to reach the maximum threshold for FXR regulation of PLTP, which could explain why we do not observe a QTL for Pltpq2 on the atherogenic diet. Sequencing of Nr1h4 found six base pair differences, leading to amino acid difference in FXR (Table II). Another candidate gene for this same QTL is the gene-encoding Apo F (Apof). Apo F is known to inhibit the phospholipid transfer activity of cholesterol ester transfer protein (CETP) in human20 and inhibits CETP in humans; it might also inhibit PLTP, another member of the lipopolysaccharide-lipid transfer protein gene family.22 We found two SNPs within the coding region of the gene resulting in nonconserved amino acid differences. One (AA128) is in the pre-protein, whereas the other (AA267), is in the part of the protein that is bound to HDL after cleavage. If Apo F is an inhibitor of PLTP, this latter substitution might be responsible for differential inhibition. Nr1h4 and Apof are located at 50 and 73 cM, on either side of the 95% confidence interval of Pltpq2. Our current hypothesis is that both genes may be involved and that we have two closely linked independent QTL in this region as suggested by the test for multiple QTL. If two QTL exist, this would also explain the seemingly strange allele effect of the QTL, where heterozygous animals for the peak marker D10Mit271 have a significantly higher PLTP activity than homozygous animals for both parental genotypes. If both genes are involved in PLTP activity, we might expect opposite allele effects for these genes, which could lead to a net allele effect at D10Mit271, as depicted in Figure 4B.
After 6 weeks of the atherogenic diet, we found different QTL associated with PLTP activity. We did not find the expected increase in Pltp mRNA expression, which might be because of compensatory mechanisms in gene regulation. We did not observe Pltpq1 and Pltpq2 on the atherogenic diet and speculate that the atherogenic diet causes the factors underlying these QTL to reach maximum levels for PLTP regulation both in NZB and SM, and therefore eliminates differences between the two strains. Instead, we found two novel QTL, Pltpq3 and Pltpq4 on chromosomes 9 and 15, respectively. Because the promotor sequence of Pltp contains sterol regulatory element-binding protein-1 (SREBP-1) binding sites,7,8 we consider the gene for SREBP cleavage-activating protein (Scap) as a candidate gene for Pltpq4. However, we found no evidence to support the candidacy of this gene because expression did not differ and the coding region contained only one basepair difference, leading to a conservative amino acid change. Ppara, whose gene product is involved in Pltp regulation might be a candidate gene for Pltpq4. Measuring the relative expression of Ppara in liver showed no significant difference between NZB and SM on the atherogenic diet. Interestingly, we do see a significant (P=0.0034) difference between NZB and SM on the chow diet, but this apparently does not influence the PLTP activity on chow enough to be detected as a QTL. Two sequence differences, one in the 5′UTR and one in the 3′UTR of the gene, were found between SM and NZB. These SNPs might influence the translation efficiency of the mRNA.
The gene for an orphan nuclear receptor SHP, Nr0b2, is a candidate for Pltiq1. Although a direct relation between SHP and PLTP has not been found, Nr0b2 is regulated by FXR23 and is known to be involved in regulation of phospholipid metabolism. We found no sequence differences in the coding sequence between the two strains. The results of the expression analysis for SHP show little difference between diets for NZB but an increase in atherogenic diet fed SM (P=0.015). If SHP regulates PLTP expression, inducibility would be smaller for NZB than for SM. However, the allele effect for D4Mit312 shows the opposite (Figure 4E). Therefore, we must conclude that Nr0b2 is probably not the gene responsible for Pltiq1 and the differences in expression are probably a direct effect of FXR levels.
In conclusion, we have identified 5 QTL that regulate PLTP activity between the two inbred mouse strains NZB and SM. Considering the relation between PLTP activity and expression levels of PLTP in the liver, it is likely that some of the QTL are transcription factors that have an expressional or functional difference between the two inbred strains. Nr1h4 is a good candidate gene for Pltpq2, the QTL on chromosome 10. The difference of both inbred strains between PLTP activity on chow and atherogenic diet cannot be explained by expression differences of Pltp. Therefore, factors other than transcription factors might underlay some of the QTL. Apo F, which is involved in CETP inhibition and has an amino acid difference between SM and NZB, is a good candidate for Pltpq2. Thus, we have found evidence for two candidate genes for Pltpq2 on chromosome 10, Nr1h4 and Apof, and some evidence that two closely linked QTL exist at this locus. Further study will be necessary to identify the genes underlying these QTL.
The authors thank Hal Kennedy, Tina Snow, Colleen Bradstreet, Eric Taylor, Susan Sheehan, and David Higgins for their technical assistance. This work was funded by PO1 HL 30086 and the Program for Genomic Applications (grant HLB 66611) from the Heart, Lung, Blood and Sleep Disorders Institute (US National Institutes of Health).
- Received September 17, 2003.
- Accepted October 21, 2003.
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