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Biology of Reproduction 61, 1216-1225 (1999)
© 1999 Society for the Study of Reproduction, Inc.

Articles

Analysis of Expression of Genes Involved in Apolipoprotein E-Based Lipoprotein Metabolism in Pregnant Mice Deficient in the Receptor-Associated Protein, the Low Density Lipoprotein Receptor, or Apolipoprotein E1

Lieve Umans4,a, Lut Overbergh3,,4,a, Lutgarde Serneelsa, Ina Tesseura, and Fred Van Leuven2,a

a Experimental Genetics Group, Center for Human Genetics (CME), Flemish Institute for Biotechnology (VIB), Katholieke Universiteit Leuven, Campus Gasthuisberg, B-3000 Leuven, Belgium


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mice deficient in receptor-associated protein (RAP) were phenotypically normal, but in contrast to results previously reported in RAP-;cl- mice, nearly 50% of the offspring died at or shortly after birth. To attempt to determine the reason for this, we analyzed the regulation of expression of genes involved in apolipoprotein E (apoE)-based mechanisms in RAP-deficient mice and compared this to results in mice deficient in low density lipoprotein receptor (LDLR) or apoE. The major finding concerned a large increase in hepatic lipoprotein receptor-related protein (LRP) mRNA and LDLR mRNA levels in pregnant RAP knockout mice. This is in contrast to the down-regulation of LRP mRNA and LDLR mRNA, which is normally seen in wild-type mice. Also in LDLR knockout mice, a significant up-regulation in expression of LRP mRNA was demonstrated. In apoE knockout mice, hepatic LRP mRNA did not change significantly, while hepatic LDLR mRNA expression was increased. In placenta and uterus, the deficiency of RAP did not markedly affect the expression of LRP and LDLR. Lipoprotein lipase mRNA and apoE mRNA increased during pregnancy in all mice, independent of their genetic status. The current study does not directly explain the increased mortality of RAP-;cl- pups. The data demonstrate, however, important relative changes in expression of the genes analyzed, an indication that LRP and LDLR play an important role in lipid metabolism during pregnancy.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In addition to low density lipoprotein receptor (LDLR) [1], the LDLR family of lipoprotein receptors comprises lipoprotein receptor-related protein (LRP) [2–4] (or alpha-2-macroglobulin-proteinase complex receptor) [5], megalin, glycoprotein 330 or lipoprotein receptor-related protein 2 (gp330 or LRP2), very low density lipoprotein receptor (VLDLR) [6], apoER2 [7], LR8B [8], and sorLA/LR11 [9, 10]. Recently, new members of the LDLR family have been characterized, i.e., LRP3 [11], LRP4 [12], LRP5 [13], and LRP6 [14]. All are evolutionarily related single membrane spanning receptors, built of structurally similar elements. Functionally, LDLR is characterized as a key player in cholesterol homeostasis, mediating uptake of lipoproteins containing apolipoprotein B and/or E [15]. In humans, this role is demonstrated clinically by a large number of different mutations in the LDLR gene that cause familial hypercholesterolemia [1].

The complexity of lipid and lipoprotein metabolism, based on a diversity of carrier proteins, modifying enzymes, and cellular receptors, makes elucidation of regulatory mechanisms in the intact animal difficult. Transgenic mice have proven to be useful models of normal and disturbed lipoprotein metabolism; i.e., mice deficient in LDLR [16–18] or apolipoprotein E (apoE) [19–21], among others, have been studied as models for atherosclerosis and related human diseases. Other engineered mouse strains of interest contain deletions of other members of the LDLR family or of their chaperon, the receptor-associated protein (RAP). It has been proposed that RAP functions as an intracellular chaperon required for folding and maturation of many endocytic lipoprotein receptors, probably by protecting binding sites during receptor biosynthesis ([22] and references therein). RAP, a 40-kDa protein, is localized mainly within the rough endoplasmic reticulum and binds with high affinity to LRP [23, 24], to megalin [25–27], and to VLDLR [28, 29] and with low affinity to LDLR [30]. Both LRP and RAP are expressed in most organs of mice; however, within each tissue the cellular distribution of LRP and RAP is quite different. The most marked extremes are observed in the kidney and placenta [31, 32].

LRP is a multifunctional receptor, binding a collection of diverse ligands, including proteinases, proteinase inhibitors and their complexes, apoE-enriched lipoproteins, lipoprotein lipase (LPL), and others (for reviews see [33–36]). In contrast to LDLR and VLDLR, LRP is essential for mouse embryogenesis, since targeted inactivation of the LRP gene is lethal in the second week of pregnancy [37]. This precludes the use of LRP-;cl- mice for analysis of LRP deficiency during pregnancy. The VLDLR binds apoE-containing lipoproteins, i.e., very low density lipoproteins (VLDL), VLDL remnants, and intermediate density lipoproteins [38, 39]. Expression of VLDLR is essentially absent in liver, while high levels are observed in various tissues including the placenta, suggesting a role in placental lipid metabolism [6, 40]. However, knockout of the VLDLR gene demonstrated only that VLDLR plays a role in adipose tissue metabolism and fat accretion [41]. Previously, we have analyzed comprehensively the expression of many of these and related genes, measuring both mRNA levels and their patterns of expression, during the physiologically important and critical periods of pregnancy and birth in wild-type mice [31, 32, 42]. In the last of these studies we established new aspects of compensatory mechanisms operating in pregnancy and at birth. The changes in expression patterns of LRP, LDLR, and LPL, as well as the large increase in apoE synthesis, demonstrated that the uptake of cholesterol and lipids in the placenta is provided by increased uptake of apoE-VLDL via LRP, as well as by increased fatty acid uptake generated by LPL overexpression. During pregnancy and at birth there is a pronounced shift toward apoE-based lipoprotein metabolism in the placenta [42].

We have now generated and characterized RAP-deficient mice and, consistent with an independent report [43], confirm that RAP-deficient mice are phenotypically normal, viable, and fertile. It has not previously been reported, however, that RAP-;cl- mice reproduce less efficiently than any of our other transgenic mouse strains when housed under identical conditions. This is due to higher mortality of RAP-deficient pups at or around birth. Therefore we analyzed the effect of RAP deficiency on the expression of lipoprotein receptors and their ligands during pregnancy, as previously performed in wild-type mice [42]. We report here the analysis of expression of the apoE receptors LRP, LDLR, and VLDLR and of their ligands apoE, LPL, and RAP in pregnant mice from strains that are deficient in RAP, LDLR, or apoE. Although these results do not explain the high mortality of the RAP-;cl- pups directly, the data presented demonstrate important relative changes in expression of the genes analyzed.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
RAP Gene Targeting

From cosmid clone pMoc1.3 [44], we subcloned an 8-kilobase (kb) XhoI fragment containing exons 3 to 8 of the mouse RAP gene. A 0.7-kb SmaI fragment containing part of exon 6 and intron 6 was replaced by a 1.8-kb BglII fragment containing a cassette encoding the hygromycin B phosphotransferase gene driven by the phosphoglycerate kinase (PGK) promoter [45, 46]. The targeting construct with the embedded hygromycin cassette was about 9.2 kb in size. After digestion with XhoI, the construct was electroporated into embryonal stem cells (ES cells, line E14) [47]. ES cells were cultured on mitomycin-treated STO fibroblast feeder layers with suitable resistance to antibiotics, allowing positive selection in medium containing hygromycin B (100 µg/ml). ES colonies were picked, expanded, frozen, and genotyped by Southern blotting with probes located outside and at either end of the targeting construct (Fig. 1): a 700-base pair (bp) genomic KpnI fragment located upstream at the 5' end (L probe); a 900-bp XhoI-SalI genomic DNA fragment located downstream of the 3' end (R probe); and a 1.8-kb BglII fragment of the PGK-hygromycin gene [45, 46, 48]. Homozygous deficient mice were obtained essentially as described previously [46]. Southern blotting on tail-biopsy DNA authenticated the mutated alleles. Routine genotyping was performed by polymerase chain reaction (PCR) on tail-biopsy DNA after digestion with proteinase K (1 mg/ml in 0.5% SDS, 0.1 M NaCl, 50 mM Tris, 1 mM EDTA, pH 8.0, at 55°C for 18 h). Proteinase K was inactivated (98°C, 10 min); the lysate was diluted in Tris-EDTA buffer (1 mM Tris, 1 mM EDTA), and aliquots were used in mixtures containing each primer at 0.1 µM, each dNTP at 200 µM, 50 mM KCl, 10 mM Tris-HCl, 1.5 mM MgCl2, and 1.25 units of Taq DNA polymerase in a total volume of 50 µl. The wild-type RAP allele was amplified with primers located in exon 6 and exon 7 (5'GGTGATAGATCTGTGGGA3' and 5'GCTGCTTCTGGTAGTGGT3'). The mutated allele was amplified with a forward primer in the promoter region of the PGK-HYG gene (5'GATGTGGAATGTGTGCGA 3') in combination with the same reverse primer located in exon 7 of the RAP gene. Amplification generated a 250-bp fragment for the mutated allele and a 1-kb fragment for the wild-type allele.

LDLR- and apoE-Deficient Mice

LDLR-;cl- mice were obtained from Jackson Laboratories (Bar Harbor, ME) [16], and apoE-;cl- mice were generously donated by M. Hofker and L. Havekes (Leiden University, The Netherlands) [21]. All knockout mice used in the presented experiments were derived from 129 ES cells and back-crossed to the C57Bl/6 strain for at least 4 generations. All mice were housed in the same room in the local animal house with free access to food and water, under a 13L:11D light cycle (lights-on at 0700 h), at a temperature of 21°C and at 60% relative humidity. The mice were regularly monitored for general health conditions and for behavioral aspects. All experimental procedures, supervised by a veterinary doctor, were performed according to university, Flemish, Belgian, European, and international legal standards and regulations.

RNA Extraction, Northern Blotting, and DNA Probes

The liver, uterus, and placenta were isolated from 3 wild-type C57Bl/6 mice, 3 LDLR-;cl-, 3 apoE-;cl-, and 3 RAP-;cl- female mice that had been pregnant for 12 and 19 days or at the day of birth. Cerebrum, cerebellum, kidney, testis, lung, spleen, heart, and muscle were isolated from wild-type mice, heterozygotes, and homozygotes. Total RNA and poly(A) mRNA were extracted, purified, separated, and blotted as described previously [31]. The cDNA probes used were mouse apoE cDNA probe (pmEUC18 obtained from S. Tajima, National Cardiovascular Center Research Institute, Suita, Osaka, Japan) [49]; the mouse LRP cDNA probe (1.4-kb EcoRI fragment, position 776-2197 in mouse LRP cDNA, [50]); LPL cDNA probe (a 1-kb PstI restriction fragment from rat LPL cDNA, provided by J. Auwerx, Institut Pasteur de Lille, Lille, France); mouse RAP cDNA probe (a PCR fragment, position 8-1078 of mouse cDNA; [51]); VLDLR cDNA probe (a PCR fragment, position 1086-2181 of mouse cDNA) [6]; and ß-actin cDNA probe (Clontech, CA).

The two transcripts observed for RAP mRNA (1.8 and 3.6 kb) [44] and for VLDLR mRNA (3.9 and 7.9 kb) [42] were always present in comparable relative amounts, indicating that they result merely from different termination points on the respective genes; they are therefore not discussed separately.

Western Blotting

For Western blotting, frozen liver tissue was mechanically homogenized in 4 ml buffer containing 10 mM Tris, 50 mM NaCl, 30 mM sodium pyrophosphate, 50 mM NaF, 200 µM Na3VO4, 1% (v:v) Triton X-100, and a mixture of proteinase inhibitors. The homogenized samples were centrifuged (27 000 x g, 35 min); samples of the supernatant were denatured by addition of SDS (2.3% final), boiled for 10 min, and reduced with 2-mercaptoethanol (1% final). After separation in linear gradient (4–20%) Tris-glycine SDS-PAGE, proteins were electrophoretically transferred to Hybond-C membranes (Amersham, Piscataway, NJ). Blocking with fat-free milk was followed by sequential incubation with rabbit antiserum (F36), then with goat anti-rabbit IgG labeled with peroxidase; the immune complexes were visualized with the enhanced chemiluminescence system (Amersham, Buckinghamshire, UK). The polyclonal antiserum (F36) reacts specifically with the 85-kDa subunit of LRP and with its 600-kDa precursor form. It was prepared by immunizing rabbits with a synthetic peptide corresponding to the carboxy terminal of the cytoplasmic domain of human LRP (residues 4513–4525) coupled to keyhole limpet hemocyanin.

Statistical Analysis

Each time point in the histograms (Fig. 2), i.e., Days 12 and 19 postcoitus (pc) and 1 day after birth (Day +1), represents the calculated mean of determinations for three individual mice. The data from the Northern blotting were analyzed by the Mann Whitney U-test, and the data from Table 1 were analyzed by the two-tailed Student's t-test. Significance was defined at the 0.05 level.


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  		TABLE 1. Breeding of homozygous mice deficient in RAP, LDLR, ApoE, MAM, or MAM and MUG, with a mixed background (C57Bl + 129/Ola) or a 99% C57Bl background (backcrossed to the C57Bl strain for 6 generations)


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Targeting of the RAP Gene

The construct used to target the RAP gene contained an 8-kb XhoI fragment comprising exons 3 to 8 of the RAP gene. A 0.7-kb SmaI fragment, containing 68 bp of exon 6 and part of intron 6, was replaced by a 1.8-kb BglII fragment encoding the hygromycin B phosphotransferase gene driven by the PGK promoter [45] (Fig. 1A). Before electroporation, the targeting sequences were excised from the vector by digestion with XhoI. Selection by inclusion of hygromycin in the medium yielded several thousand colonies; 176 of these were genotyped by Southern blotting with probe R (Fig. 1), resulting in 18 candidate cell lines that were expanded. Final selection was based on genotyping by Southern blotting after EcoRV or EcoRI digestion and sequential hybridization with the three DNA probes R, L, and HYG (Fig. 1). Eight ES cell lines with a correctly targeted RAP allele were injected into C57Bl/6 blastocysts, and a chimera that transmitted the targeted RAP gene effectively through the germ line was selected to found the RAP-deficient mouse strain.



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  		FIG. 1. Inactivation of the RAP gene in ES cells. A) Schematic representation of the targeting vector, the wild-type murine RAP gene, and the targeted RAP gene as described in the text. Exons are represented by black boxes and numbered as described previously [36]. The hatched box marked HYG denotes the hygromycin selection marker. Restriction sites: XhoI denoted by X, EcoRI by E. Short bold lines marked R and L represent the DNA probes used in genotyping by Southern blotting (see Materials and Methods). Homologous recombination resulted in loss of the wild-type 16-kb EcoRI fragment and generated EcoRI fragments of 6.5 kb detected by the R probe and 10 kb detected by the L probe and the HYG probe, which also reacted with the 0.6-kb fragment as indicated. Triangles indicate the location of synthetic oligonucleotide primers used in PCR amplification (see text for details). B) Genotyping of genomic DNA of positive ES cell lines, by Southern blotting, digested with EcoRI and hybridized with the three DNA probes R, L, and HYG as indicated. Diagnostic bands of 6.5 kb (R probe) and 10 kb (L probe and HYG probe) are shown, relative to radiolabeled DNA markers (lane MW). C) Northern blot of total RNA isolated from cerebrum, cerebellum, liver, and kidney of homozygous (lane 1), heterozygous (lane 2), and wild-type mice (lane 3), hybridized with probes specific for RAP [36, 43] and actin as indicated. The typical 1.8-kb and 3.6-kb RAP mRNA species were detected. Note the presence of the cross-reacting mRNA species of about 1.6 kb that was detected in the kidney of RAP-;cl- mice (see text for more details). D) Western blot of protein extracted from liver, cerebrum, and kidney of RAP-;cl- mice (panel 1), RAP+;cl- mice (panel 2), and wild-type mice (panel 3), probed with a specific rabbit antiserum [44]. The molecular weight of marker proteins is indicated on the left (x 10-3)

The effectiveness of RAP gene inactivation was demonstrated by analysis of RAP mRNA and protein. Total RNA was isolated from cerebrum, cerebellum, liver, kidney, lung, spleen, testis, heart, and muscle of wild-type, heterozygous, and homozygous RAP-deficient mice. Northern blotting using RAP cDNA as probe revealed the absence of the two RAP mRNA transcripts of 1.8 and 3.6 kb (Fig. 1C) [31, 44, 51]. In all tissues from heterozygous mice analyzed, the level of RAP mRNA was between 40% and 60% of that in wild-type animals (Fig. 1C). In the kidney, which contains the highest levels of RAP mRNA in wild-type mice [31], a weakly reacting smaller mRNA species of about 1.6 kb was detected in homozygous RAP-;cl- mice (Fig. 1C). Western blotting with a specific antiserum [52] on extracts from major organs never detected full-length RAP protein, but a cross-reacting protein of less than 30 kDa was detected in kidney of homozygous RAP-deficient mice (Fig. 1D). Presumably, this protein represents either a truncated RAP protein or a cross-reacting, related protein that is up-regulated in RAP-;cl- mice. This is currently being investigated.

Phenotype of RAP-;cl- Mice

Initially six breeding pairs of heterozygous RAP+;cl- mice yielded a total of 99 pups that were genotyped by Southern blotting. In this cohort, 18% of the mice were homozygous wild type, 48% were heterozygous, and 33% were homozygous for the targeted RAP allele. Clearly, RAP deficiency did not adversely affect Mendelian inheritance or the fertility of heterozygous RAP+;cl- mice. Surprisingly, subsequent breeding of homozygous RAP-deficient mice into the C57Bl/6 genetic background resulted in litters of a small size. This was significantly different from results for RAP-;cl- mice with a mixed background (C57Bl + 129/ola) (P = 0.001) and from those for nearly all the other knockout mouse strains analyzed (P < 0.001) (Table 1), with the exception of LDLR-;cl- mice (P = 0.07). MAM-;cl- [46] and MAM-;cl-MUG-;cl- mice [53] (alpha-2-macroglobulin [MAM] and murinoglobulin-1 [MUG] deficient) with a C57Bl background, also generated in our laboratory, raised significantly more pups compared to RAP-;cl- C57Bl mice (P < 0.001).

Adult RAP-;cl- mice remain healthy and active, without any obvious signs of disease for up to 2 yr. As stated above, however, the RAP-;cl- strain continued to be less prolific than expected, an observation not reported previously [43] (Table 1). RAP-;cl- mice back-crossed into the C57Bl/6 background for 4 generations and more yielded a normal number of litters, but, compared to litters from the other knockout mice strains, these contained significantly fewer pups (P < 0.002); in addition, fewer pups (P < 0.001) survived the first postnatal days (Table 1). The difference in survival rate between RAP-;cl- C57Bl and LDLR-;cl- C57Bl was not significant (P = 0.07), due to the limited number of LDLR-;cl- litters, although a trend for a higher mortality rate for RAP-;cl- C57Bl pups relative to LDLR-;cl- pups was noted (Table 1). The litter size of RAP-deficient mice before birth (embryonal Day 18) was normal compared to that of wild-type mice (Table 2), indicating lethality during the last days of pregnancy or at birth. None of the 1-day-old pups showed obvious defects; thus it was not possible to predict which pups would die or which would be killed by the mother. Usually, dead pups were quickly eaten by the female, which prevented their examination. Although mortality rates in the initial breeding (RAP-;cl- C57Bl + 129/ola) seemed normal, statistical analysis demonstrated (Table 1) significant differences between these mice and MAM-;cl- mice (P < 0.001) and MAM-;cl-MUG-;cl- mice (P < 0.001) with the same mixed background. Comparison of the number of surviving pups of MAM-;cl- and MAM-;cl-MUG-;cl- mice with a mixed background and with a homogenous C57Bl background indicated that also in these mice the C57Bl background decreased the number of surviving pups (P < 0.01). These data demonstrate that the C57Bl background and RAP deficiency together are responsible for the smaller litter size and the low survival rate. Therefore, as one line of approach to this problem, we analyzed the expression patterns of genes encoding LRP, LDLR, and VLDLR, lipoprotein receptors to which RAP binds [23, 24, 28–30], and genes encoding their ligands apoE and LPL, in RAP-;cl- mice. In parallel, LDLR-;cl- and apoE-;cl- mice were included in the analysis to extend our previous studies and to gather additional information on apoE-based lipoprotein metabolism during pregnancy [42].


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  		TABLE 2. Litter size of RAP-/- mice and wild-type mice before birth (embryonal day 18)

Analysis of Levels of mRNA Coding for LRP, LDLR, VLDLR, RAP, apoE, and LPL

Levels of mRNA encoding lipoprotein receptors LRP, LDLR, VLDLR, their chaperon RAP, and their ligands apoE and LPL were analyzed by Northern blotting during pregnancy and at birth in females of the wild-type C57Bl;cl6 mouse strain and mice deficient in either RAP, LDLR [16], or apoE [21]. Hepatic VLDLR mRNA was not examined since expression of VLDLR is essentially absent in the liver [6, 40]. Total liver and uterus RNA was extracted at Days 12 and 19 pc and at term (denoted Day +1), while placental RNA was isolated at Days 12 and 19 pc. The rationale for this approach was based on previous findings in pregnant wild-type mice [42]. The current experiments were aimed at detecting changes in expression of the genes concerned, by comparison of their mRNA levels in the 3 different organs in the 4 different mouse strains at the different time points during pregnancy and at birth. For clarity, the resulting data are presented in histogram format (Fig. 2). The most relevant findings for each knockout mouse strain are described below, in the context of results previously obtained in wild-type mice [42].



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  		FIG. 2. Quantitation of mRNA coding for LRP, LDLR, VLDLR, RAP, apoE, and LPL in the liver, placenta, and uterus of wild-type, RAP-;cl-, LDLR-;cl-, and apoE-;cl- mice. Sequential hybridization of Northern blots with probes specific for the specified mRNA were quantitated by densitometric scanning, and results (mean + SE) are expressed in relative units after normalization for different loading of mRNA by hybridization for actin (see also Fig. 1). All panels are marked with the specified genetic background of the mice, the specified organ, and the mRNA species analyzed, with alternative splice forms indicated where appropriate (see text for more details). Data is shown in shaded boxes for Day 12 pc, dark boxes for Day 19 pc, and open boxes for Day +1. Each time point represents the calculated mean of determinations for three individual mice

RAP Knockout Mice (RAP-;cl- Mice)

The proposed function of RAP as a chaperon of lipoprotein receptors [22, 54] led us to anticipate that its absence would, additionally, place stress on the system of apoE-mediated lipoprotein transport via LRP during pregnancy, which has been previously documented in wild-type mice [42]. In addition, these RAP-;cl- mice can provide interesting information regarding the role of LRP during pregnancy, since the embryonal lethality caused by targeted inactivation of the LRP gene [37] prevents direct analysis of the influence of LRP deficiency.

The collected data, however, did not explain the high mortality of RAP-;cl- offspring around birth. Indeed, no marked changes in mRNA levels were noted in the placenta and uterus of pregnant RAP-;cl- mice relative to wild-type mice (Fig. 2). The major difference was a considerable increase of LRP mRNA (P < 0.02) and LDLR mRNA levels (P < 0.03) in the liver of RAP-;cl- females toward delivery. This is in contrast to observations in wild-type mice, which were characterized by a 4-fold reduction in hepatic LRP mRNA levels (P < 0.04) in concert with a 2-fold decrease in hepatic LDLR mRNA (P < 0.03). Similarly to findings in wild-type mice, increases in expression of apoE mRNA in placenta and uterus were much more pronounced than in liver (Fig. 2).

Since RAP influences processing and maturation of LRP [22, 43], an attempt was made to determine and correlate the relative changes of the 600-kDa LRP precursor protein and of the 85-kDa LRP processed subunit in the liver of wild-type and RAP-;cl- mice. Western blotting was performed using an antiserum specific for the C-terminus of LRP [50]. The level of the 600-kDa precursor of LRP was significantly increased in the liver of RAP-;cl- mice relative to wild-type mice at Day 19 pc and Day +1 (P < 0.001) (Fig. 3). At the same time points, the level of the 85-kDa subunit was significantly decreased (P < 0.01) (Fig. 3). In nonpregnant females it has already been demonstrated that in the absence of RAP, maturation is slow, resulting in accumulation of the uncleaved 600-kDa precursor [22, 43, 55]. The 4-fold increase in LRP mRNA level in the liver of pregnant RAP-;cl- mice from Day 12 pc to Day +1, however, did not result in higher levels of mature LRP protein (Figs. 2 and 3). On the contrary, the amount of the 85-kDa LRP subunit on Day 19 pc and Day +1 was significantly decreased compared to that on Day 12 pc (P < 0.01) (Fig. 3).



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  		FIG. 3. Quantitation of LRP protein in liver of RAP-;cl- and wild-type mice. Levels of LRP 600-kDa precursor and of the 85-kDa processed subunit (which is equimolar to the 515-kDa subunit) were measured by densitometric scanning of Western blots with the F36 antiserum (see section on experimental procedures). Extracts of liver of RAP-;cl- mice (white boxes) and wild-type mice (black boxes) at the indicated time points, i.e., Day 12 pc, Day 19 pc, and Day +1 after birth, were analyzed. Each histogram represents the mean of determinations for three individual mice, and each blot was subsequently probed with antiserum to mouse actin to allow quantitation and normalization

LDLR Knockout Mice (LDLR-;cl- Mice)

Unexpectedly similar to findings in RAP-;cl- mice and again opposite to those in wild-type mice, levels of the 15-kb LRP mRNA increased significantly toward delivery in the liver of pregnant LDLR-;cl- mice (P < 0.05) (Fig. 2) [42]. The timing was different, however, since in RAP-;cl- mice the hepatic LRP mRNA reached maximal levels by Day 12 pc, while in LDLR-;cl- mice the maximum levels were detected at birth (Figs. 2 and 4). Since in the liver of wild-type females the level of LRP mRNA decreases 4 times and hepatic LRP mRNA in LDLR-;cl- females increases 6 times, the liver of LDLR-;cl- female mice contained relatively 24 times more LRP mRNA than the liver of wild-type females at delivery (Fig. 4). In contrast to observations in the uterus of wild-type mice, where the level of LRP mRNA did not change noticeably, there was a trend for up-regulation of the level of LRP mRNA in the uterus of pregnant LDLR-;cl- females, although not significant (P < 0.06) (Fig. 2). The placental regulation of expression of the apoE receptors in LDLR-;cl- mice, on the other hand, was very similar to that in wild-type mice (Figs. 2 and 5).



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  		FIG. 4. Northern blot of hepatic LRP mRNA from pregnant wild-type and LDLR-;cl- mice. Hepatic mRNA was probed sequentially for LRP mRNA (15 kb) and for actin mRNA (2 kb) for normalization. RNA was extracted from wild-type mice (C57Bl) and from LDLR-;cl- mice at Day 12 pc, Day 19 pc, and Day +1 as specified

The hepatic up-regulation of LRP mRNA in LDLR-;cl- mice was accompanied by a significant increase in expression of RAP (P < 0.03) and apoE (P < 0.03) mRNA and only a moderate increase in LPL mRNA (P = NS). Also noted was a very pronounced increase in apoE (P < 0.03) and LPL mRNA (P < 0.03) in the placenta of LDLR-;cl- mice; this value, rising about 30-fold at Day 19 pc, was already increased about 10-fold in pregnant wild-type mice. This massive increase in apoE and LPL mRNA was a general observation in the placenta and uterus in all mice analyzed, although less pronounced in the other groups than in the LDLR-;cl- mice.

ApoE Knockout Mice (ApoE-;cl- Mice)

Data from the literature and results previously obtained in wild-type mice ([42] and references therein) demonstrated that apoE-mediated lipoprotein import in the placenta of females in late pregnancy is a physiologically important mechanism. We questioned therefore how apoE-deficient mice cope in this respect, since they are reproductively not impaired or hindered [20, 21], in contrast to RAP-;cl- mice. The findings were, however, not informative in this respect, since the documented changes in mRNA levels in pregnant apoE-;cl- mice were rather similar to those in wild-type mice (Fig. 2), with only some differences. The most remarkable effect was the increase of hepatic LDLR mRNA levels (P < 0.03) toward delivery, similar to that in RAP-;cl- mice and opposite to the decrease in hepatic LDLR mRNA levels in wild-type mice. The increase in hepatic LRP mRNA observed in RAP-;cl- mice, however, was not demonstrated in apoE-;cl- mice (Fig. 2). This was interpreted to mean that in the absence of apoE, lipoprotein import via LRP was not affected or even compromised and was compensated for by increased expression of LDLR mRNA.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We generated mice lacking functional RAP protein in order to study the physiological role of this chaperon in vivo. The resulting RAP-deficient mice were fertile and devoid of marked phenotypic abnormalities, confirming a published report [43]. Previously unreported was the observation that homozygous RAP-;cl- mice reproduce less efficiently than expected, despite being normally fertile as judged by mating with wild-type partners. The decreased litter size is due to high mortality of RAP-;cl- pups at or shortly after birth, specific for this strain and not caused by gross malformations or by environmental parameters. Statistical analysis demonstrated that both the C57Bl background and deletion of the RAP gene significantly decreased the number of pups and the survival rate, indicating that not only the RAP gene but also the strain background influences reproduction. In this study we investigated the effect of RAP deficiency on apoE-based lipoprotein metabolism during pregnancy. We analyzed the levels of expression of mRNA encoding LRP, LDLR, VLDLR, RAP, apoE, and LPL, which are all genes involved in lipoprotein metabolism, in liver, uterus, and placenta from pregnant females and at birth. Expression levels in RAP-;cl- mice and in mice deficient in LDLR or apoE were compared to those in C57Bl wild-type mice (see also Introduction). The approach was based on previous studies in wild-type mice demonstrating that pregnancy caused an important and generalized shift to apoE-based lipid metabolism to provide nutrients and lipids to the fetus ([42] and references therein).

The current study does not directly explain the increased mortality of RAP-;cl- pups but has provided new and important clues to the compensatory mechanisms that are operating at the level of gene expression. These include important changes in expression of RAP mRNA itself and of LRP as the functionally most closely associated receptor. The major finding concerned the large increase in LRP mRNA levels, and to a lesser extent the increase in LDLR mRNA, in the RAP knockout mice in contrast to the decrease in LRP mRNA and LDLR mRNA levels usually seen at the same time points in wild-type mice. This reversal in regulation of expression of LRP and LDLR was also evident in LDLR-;cl- mice. In apoE-;cl- mice, this reversal was observed only for LDLR. Since both apoE and LDLR knockout mice reproduced normally, this difference in regulation of expression can be excluded as the cause of mortality.

The comparative analysis of hepatic levels of LRP mRNA and protein in RAP-;cl- mice relative to wild-type mice revealed that posttranslational regulation and/or increased turnover of LRP protein could explain this difference. The increase in LRP mRNA was not reflected by an increase in mature LRP protein, probably because of a decrease in the rate of the processing and maturation of LRP protein in the absence of RAP. We postulate that the increase in LRP mRNA is in reaction to the lack of functional expression of the LRP protein in pregnant RAP-;cl- mice. This can be verified experimentally by measuring rate of transcription of the LRP gene, LRP mRNA turnover and stability, and rate of translation and turnover of LRP precursor and processed subunits. However, these measures were outside the scope of the current study. Such an examination will lead to an understanding of the molecular signaling mechanisms by which LRP and other members of the LDLR family regulate their functional expression at the cell surface in normal conditions, as well as in pregnancy, at birth, and in disease.

The results shed important new light on the physiological mechanisms that operate during pregnancy at the level of tissues and overall in vivo. As demonstrated before [42], during pregnancy there is a down-regulation of LRP and LDLR mRNA in the liver, which decreases the capacity of LDLR and LRP to take up lipoproteins. As a consequence, the levels of circulating lipoproteins are expected to increase, chylomicrons and VLDL are subjected to hydrolysis by LPL, and the fatty acids generated can freely cross the placenta. This response is completely reversed in RAP-;cl- and LDLR-;cl- mice and to some extent also in apoE-;cl- mice. The lack of either RAP or LDLR thus overrules the (unknown) signals that normally operate during pregnancy in wild-type mice, and increased expression of LRP could compensate for the decreased levels of LDLR in maintaining lipid homeostasis. It should be noted that this is the normal situation in the placenta, as placental LRP mRNA increased 5- to 10-fold toward term in all pregnant mice analyzed, independent of their genetic makeup. Increased LRP mRNA was always accompanied by similar increases in RAP mRNA, both in liver and placenta, indicating that the chaperon is mobilized to functionally support increased expression of LRP. Nevertheless, this support appeared as not essential in RAP-;cl- mice and is not absolutely required. This indicates that the exact physiological role of RAP in vivo could be outside the present paradigm.

In the placenta, apoE synthesis and ensuing enrichment of circulating lipoproteins are a major source of lipid import, in addition to LPL-mediated hydrolysis of lipids and delivery of free fatty acids to the placenta. The two mechanisms are not necessarily fundamentally different, since the binding of LPL to lipoproteins and to LRP, eventually catalyzed or modulated by cell-surface heparan sulfate, might contribute [56, 57]. Despite the presumed importance of apoE-based lipid transport during the late stages of pregnancy, apoE itself is evidently also dispensable as a constituent of lipoprotein particles, and, in addition, apoE-;cl- mice reproduced normally in external conditions that were identical to those for RAP-;cl- mice. Consequently, alternative routes for supplying lipid nutrients must be operating to fully sustain placental and fetal needs, even in the complete absence of apoE. In this situation, LDLR is a prime candidate, but apolipoproteins other than apoB might substitute for apoE in mediating transport via LRP. Although in the current study we were unable to analyze double-knockout mice, i.e., mice deficient in both apoE and in LDLR, the fact that these mice are viable and reproduce normally [18] would support the last assumption.

In conclusion, the available data specify in what respect RAP is contributing to the normal mechanism of lipoprotein transport and metabolism in mice during pregnancy and at birth, but do not provide a direct cause for the mortality of RAP-;cl- offspring. Despite the large changes in levels of the different mRNA species analyzed, RAP deficiency probably has a determining influence on the protein level. We confirmed that also in liver of pregnant RAP-;cl- females, proteolytic processing and maturation of LRP are significantly reduced compared to the values in wild-type mice [22, 43]. Furthermore, it remains possible that the increased mortality of the RAP-;cl- pups must be sought outside the present paradigm. In this respect, the consequences of RAP deficiency have also been studied in brain and in primary neurons, with particular reference to maturation and processing of the LRP precursor [55]. After this study was concluded, it was demonstrated that RAP, or at least deficiency of RAP, might exert a role in modulation of somatostatin expression and function [58]. Since expression of growth hormone, somatostatin, and growth hormone-releasing hormone is tightly regulated during pregnancy [59], this would point to a new direction for exploring the current findings. The results as presented yield insight into the complexity of the regulation of lipoprotein metabolism in pregnancy and have identified several new entry points into this important problem.



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  		FIG. 5. Northern blot of placental mRNA probed for LRP, LDLR, VLDLR, RAP, apoE, and LPL. Blots with placental mRNA extracted at Days 12 and 19 pc from pregnant females of the specified genetic background were consecutively probed for LRP, LDLR, VLDLR, RAP, apoE, LPL, and actin. The size of the respective mRNA is indicated on the right in kilobases. Exposure times were different and dependent on the actual signal; e.g., the 3.6-kb RAP mRNA splice form and the 3.9-kb VLDLR mRNA transcript were much weaker than the 1.8-kb and 7.9-kb transcripts, respectively


   ACKNOWLEDGMENTS
 
We thank M. Hofker and L. Havekens, Leiden, for generously providing apoE knockout mice; N.A. Taylor for critical reading of the manuscript; and K. Crassaerts, P. Holemans, and N. Caluwaerts for expert technical assistance.


   FOOTNOTES
 
1 This investigation was supported by the ‘Fonds voor Wetenschappelijk Onderzoek-Vlaanderen’ (FWO), by NFWO-Lotto, by the Interuniversity-network for Fundamental Research (IUAP) of the Belgian Government, by the Special Biotechnology Program of the Flemish government (IWT/VLAB/COT-008). L.U. and L.O. were postdoctoral research fellows of the Special Research Fund of the K.U.Leuven. Back

2 Correspondence. FAX: 32 16 3458 71; fredvl{at}med.kuleuven.ac.be Back

3 Current address: Laboratory of Experimental Transplantation, Campus Gasthuisberg O&N 08, KULeuven, B-3000 Leuven, Belgium. Back

4 These authors contributed equally to this work. Back

Accepted: June 17, 1999.

Received: December 2, 1998.


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