(Received for publication, December 13, 1995; and in revised form, March 1, 1996)
From the
The plasma cholesteryl ester transfer protein (CETP) mediates the transfer of cholesteryl ester from high density lipoprotein to other lipoproteins. The human CETP gene produces two forms of mRNA, with or without exon 9 (E9)-derived sequences. To study the function and regulation of alternative splicing of the CETP gene, transgenic mice were prepared 1) with the metallothionein (mT) promoter driving an E9-deleted construct (mT.CETP(-E9) transgene), and 2) with the natural flanking regions (NFR) controlling expression of genomic sequences which permit alternative splicing of E9 (NFR.CETP(±E9) transgene). With zinc induction, the mT.CETP(-E9) transgene gave rise to abundant E9-deleted CETP mRNA in liver and small intestine, but only relatively small amounts of E9-deleted protein were found in plasma. The E9-deleted form of CETP was inactive in lipid transfer and produced no changes in plasma lipoprotein profile. The NFR.CETP(±E9) transgene gave rise to full-length (FL) and E9-deleted forms of CETP mRNA in liver and spleen. In response to hypercholesterolemia induced by diet and breeding into an apoE gene knock-out background, the FL CETP mRNA was induced more than the E9-deleted mRNA, resulting in a 2-fold increase in ratio of FL/E9-deleted mRNA. The expression of CETP mRNA was found to be developmentally regulated. In NFR.CETP(±E9) transgenic mice CETP mRNA levels were markedly increased in the liver and small intestine in the perinatal period and decreased in adult mice, whereas CETP mRNA in the spleen was low in perinatal mice and increased in adults. The developmental increase in CETP mRNA in the liver and spleen was preceded by an increased ratio of FL/E9-deleted forms. Thus, the E9-deleted mRNA appears to be poorly translated and/or secreted, and the cognate protein is inactive in lipid transfer and lipoprotein metabolism. CETP gene expression was found to be highly regulated in a tissue-specific fashion during development. Increased CETP gene expression during development or in response to hypercholesterolemia is associated with preferential accumulation of the full-length CETP mRNA.
The plasma cholesteryl ester transfer protein (CETP) ()is a hydrophobic glycoprotein (476 amino acids, M
74,000) that mediates the transfer and exchange
of neutral lipids and phospholipids between the plasma lipoproteins
(Tall, 1995). CETP mediates the transfer of cholesteryl ester from HDL
and LDL to triglyceride-rich lipoproteins, as a result of an exchange
of cholesteryl ester for triglyceride. The subsequent action of hepatic
lipase on triglyceride-enriched HDL particles promotes the formation of
smaller HDL species (Barter and Rye, 1994; Deckelbaum et al.,
1986) that may be optimal mediators of cellular cholesterol efflux
(Fielding et al., 1994) and optimal substrates for the
lecithin:cholesterol acyltransferase reaction (Kunitake et
al., 1992). Thus, the activity of CETP may lead to enhanced efflux
of cell membrane cholesterol into HDL and facilitation of reverse
cholesterol transport. Mice express very low levels of plasma
cholesteryl ester transfer activity, and the introduction of a human
CETP transgene results in decreased plasma HDL cholesterol levels,
formation of smaller HDL species, and stimulation of cholesteryl ester
formation in the plasma lipoproteins (Agellon et al., 1991).
In CETP transgenic mice with hypertriglyceridemia the HDL size changes
are profound (Masucci-Magoulas et al., 1996), and the mice
show decreased early atherosclerotic lesions in response to a high
cholesterol diet (Hayek et al., 1995).
The human CETP gene consists of 16 exons encompassing 25 kilobase pairs of genomic DNA (Agellon et al., 1990). The CETP mRNA is primarily expressed in the liver, spleen, small intestine, adrenal, and adipose tissue (Jiang et al., 1991; Drayna et al., 1987). There is a similar pattern of mRNA expression in transgenic (Tg) mice expressing a human CETP minigene under the control of the natural flanking sequences of the CETP gene (Jiang et al., 1992). In response to a high cholesterol diet, CETP gene transcription is increased leading to increased mRNA levels in the liver and in peripheral tissues. Hepatic CETP mRNA is also markedly increased in response to endogenous hypercholesterolemia when the CETP transgene is crossed into apoE or LDL receptor gene knock-out backgrounds (Masucci-Magoulas et al., 1996). Thus, CETP appears to be a key regulated component of reverse cholesterol transport.
In all human tissues expressing the
CETP gene, two species of mRNA are detected: a full-length form, giving
rise to active M 74,000 plasma CETP, and a shorter
variant in which exon 9-derived sequences have been removed as a
cassette (Inazu et al., 1992). The E9-deleted CETP mRNA is
formed by alternative splicing of the CETP gene transcript. E9 encodes
180 nucleotides or 60 amino acids in the central portion of the CETP
sequence. Cell transfection of the E9-deleted cDNA gives rise to a
shortened, poorly secreted protein which seems to be inactive.
Co-transfection of full-length and E9-deleted cDNAs resulted in
inhibition of the secretion of full-length, active CETP (Quinet et
al., 1993). These studies have suggested that alternative splicing
of the CETP gene may modulate the formation of active plasma CETP, and
that the E9-deleted protein may have a dominant negative effect on
expression of the full-length protein. Alternatively, the E9-deleted
CETP variant might have an occult function in vivo, not
detectable by expression in cultured cells. In this study we have used
transgenic mice to study the function and possible regulation of
alternative splicing of the CETP gene.
Figure 1: Preparation of transgenes. Transgene 8 (mT.CETP(-E9)) was derived from the metallothionein-CETP transgene (Agellon et al., 1990) by exchanging an EcoRV fragment derived from the E9-deleted CETP cDNA with the corresponding fragment of the mT-CETP transgene. Transgene 12 (NFR.CETP(±E9)) was prepared from the NFR-CETP transgene (Jiang et al., 1992) as described under ``Materials and Methods.''
Transgenic mice were prepared by microinjection of DNA into C57Bl/6
CBA/J mouse embryos. Screening was performed on tail-tip DNA by
polymerase chain reaction and Southern blotting, using standard
procedures. Digestion of genomic DNA with EcoRV yielded two
different sized fragments for mT.CETP(-E9) and NFR-CETP
transgenic mice, allowing these transgenes to be differentiated in
crosses to develop compound mice. For phenotypic characterizations of
control, transgenic and compound mice, littermates were compared. To
study the effects of hypercholesterolemia, NFR.CETP(±E9) Tg mice
were fed a high cholesterol, high saturated fat diet (1% cholesterol,
20% saturated fat) or crossed into the apoE0 background (Plump et
al., 1992; Zhang et al., 1992).
In this study we have developed two new lines of CETP Tg mice. The first line contained an E9-deleted CETP transgene under the control of the inducible metallothionein (mT) promoter (mT.CETP(-E9) Tg mice or transgene 8 (Fig. 1). This line was used to determine the effects of the E9-deleted CETP on the plasma lipoproteins. To investigate the regulation of alternative splicing, we also prepared Tg mice expressing a natural flanking region CETP transgene containing genomic sequences that provide the option to exclude E9 sequences (NFR.CETP(±E9) Tg mice; transgene 12 in Fig. 1). This line contrasts with the previously described CETP Tg mice (Jiang et al., 1992), in which cDNA was used to prepare the central portion of the minigene and thus only transcripts containing E9 are made (NFR.CETP(+E9) Tg mice or line 5203). To test a possible dominant negative effect of E9-deleted CETP on the expression of the full-length CETP (Quinet et al., 1993), we bred mice containing both mT.CETP(-E9) and NFR.CETP(+E9) transgenes. In these compound mice, we anticipated that zinc induction would increase the formation of E9-deleted CETP, but not the full-length CETP.
Figure 2: RNase protection analysis of full-length and exon 9-deleted mRNA in two lines of mice expressing the mT.CETP(-E9) transgene (Tg8, lines 1 and 2), compound NFR.CETP(+E9)/mT.CETP(-E9) transgenic mice (5203/Tg8), and nontransgenic mice (nTg). Total RNA (100 µg) was analyzed by RNase protection assay, as described under ``Materials and Methods.'' After digestion with RNase T2 the RNA was analyzed on a 5% denaturing sequence gel and subjected to autoradiography. FL and E9-deleted forms were identified by their mobility relative to standards, prepared from a stable Chinese hamster ovary cell line expressing FL and E9-deleted CETP cDNAs. All animals were treated with 25 mM zinc sulfate in their drinking water for 1 week prior to sacrifice. Note that for unknown reasons the FL and E9-deleted mRNAs sometimes appear as a doublet in the RNase protection assay; in addition, in this assay there was a nonspecific band between the FL and E9-deleted mRNAs, also seen in the nontransgenic mice.
Liver homogenates
were analyzed by immunoprecipitation with CETP monoclonal antibody TP2,
which recognizes a C-terminal epitope present in both full-length and
E9-deleted CETP (Swenson et al., 1989). The E9-deleted protein
was not detectable in the basal state, but appeared as a sharp band of M 54,000 after zinc induction (Fig. 3, Tg8) (Quinet et al., 1993). In the compound mice in
the basal state, the FL form of CETP was seen as a faint M
70,000 band, comigrating with pure recombinant
CETP (Fig. 3, FL), and the E9-deleted protein was
undetectable. With zinc induction, the E9-deleted protein appeared and
the full-length form could not be detected. Thus, the E9-deleted mRNA
gives rise to the cognate protein, but apparently not in amounts
corresponding to the massive accumulation of mRNA. The disappearance of
the FL form in induced compound mice could suggest altered processing
in the presence of the E9-deleted variant.
Figure 3:
Immunoprecipitation of CETP from liver
homogenates of transgenic mice. Homogenates of perfused liver were
immunoprecipitated with TP2-Sepharose, then analyzed by
SDS-polyacrylamide gel electrophoresis, Western blotting with I-TP2 and autoradiography. Samples are from mice
expressing the mT.CETP(-E9) transgene alone (Tg8), or
compound NFR.CETP(+E9)/mT.CETP(-E9) mice (5203/Tg8), receiving metal-free drinking water, or water
supplemented with 25 mM zinc sulfate. The FL and E9 protein
forms were determined by comparison with molecular weight standards
(not shown) and samples of pure recombinant CETP (rCETP).
In order to detect the
different forms of CETP in plasma, CETP was immunoprecipated using
monoclonal antibody TP2 (Fig. 4) and quantitated by RIA (Table 1). In mice expressing the NFR.CETP(+E9) transgene,
the FL form of CETP appeared as a doublet of M 70,000 (Fig. 4, 5203). In mT.CETP(-E9) Tg
mice, the E9-deleted form of CETP was also detected in plasma as a
doublet at M
54,000 (Fig. 4, Tg8).
The protein doublets arises from alternative N-glycosylation
of a site outside E9 (Tall, 1995). In the compound mice, both forms of
CETP were present. With zinc induction, the E9-deleted form was
increased, while the FL form was unchanged (Fig. 4).
Accordingly, after zinc induction, total CETP mass in plasma was higher
in the compound mice, compared to mice expressing each transgene alone (Table 1). There was no detectable cholesteryl ester transfer
activity in mT.CETP(-E9) Tg mice, before or after zinc induction.
CETP activity in plasma of NFR.CETP(+E9) Tg mice was similar to
previous reports (Jiang et al., 1993) and was not changed by
expression of the E9-deleted transgene in the zinc-induced compound
mice. Thus, the E9-deleted CETP was inactive. However, there was no
evidence for a dominant negative effect of the E9-deleted CETP on the
plasma levels or activity of full-length CETP.
Figure 4: Immunoprecipitation of CETP from plasma of transgenic mice. Plasma (70 µl) was incubated with TP2-Sepharose, then subjected to SDS-polyacrylamide gel electrophoresis, Western blotting with TP2, and enhanced chemiluminescence assay using sheep anti-mouse IgG (peroxidase-linked F`(ab)2. For further details see ``Materials and Methods'' and the legend to Fig. 3.
The distribution of the different forms of CETP in plasma was assessed by native polyacrylamide gradient gel electrophoresis followed by Western blotting with TP2 (Fig. 5). In contrast to binding of full-length CETP to HDL in NFR.CETP(+E9) Tg mice (Fig. 5, line 5203), in the mT.CETP(-E9) Tg mice the E9-deleted CETP gave rise to a faint diffuse smear running above the HDL (Fig. 5, Tg8). Similar patterns were previously observed with site-directed CETP mutants which bind HDL poorly (Jiang et al., 1995). This suggests that E9-deleted CETP does not bind to HDL efficiently.
Figure 5: Distribution of CETP in plasma lipoproteins as determined by native polyacrylamide gradient gel electrophoresis and Western blotting using radiolabeled TP2. Plasma (70 µl) from zinc sulfate-treated mice was electrophoresed on 4-20% native gels. The HDL region was identified in parallel lanes stained with Oil Red O.
Figure 6: Plasma lipoprotein cholesterol distribution as determined by fast-protein liquid chromatography. Plasma samples (200 µl) were analyzed on a Pharmacia Superose 6HR column, collected in microtiter plates and cholesterol content was determined by adding cholesterol reagents (see ``Materials and Methods'') to wells followed by absorbance measurement at 490 using a microtiter plate reader. The profiles show the relative content of cholesterol in the different fractions. The column was calibrated with human lipoprotein samples. VLDL eluted in fractions 11-13, LDL in fractions 15-19, and HDL in fractions 22-26.
Thus, despite massive mRNA accumulation, the E9-deleted form of CETP appears in plasma in relatively small amounts, is inactive in lipid transfer, binds HDL poorly, and has no apparent influence on the plasma lipoprotein profile. Subsequent studies were thus carried out with mice expressing an NFR.CETP(±E9) transgene to see if factors increasing CETP gene expression also increased the ratio of FL to E9-deleted CETP mRNA.
Figure 7: Changes in CETP mRNA during development. RNA was obtained from pooled embryos and newborn or adult mice (three to four mice for each time point) and analyzed by RNase protection assay. Day 0 represents birth.
To see if hypercholesterolemia altered the splicing of E9, the NFR.CETP(±E9) Tg mice were fed a high fat, high cholesterol diet. The transgene was also bred into the apoE0 background. There was a small nonsignificant increase in hepatic FL CETP mRNA in response to the high cholesterol diet or the apoE0 background (Table 3). This response was less pronounced than that obtained in other lines of NFR-CETP Tg mice, such as 5203, probably reflecting positional effects (Jiang et al., 1992). However, when compound NFR.CETP(±E9) Tg/apoE0 mice were placed on the high cholesterol diet, there was a marked induction of the full-length CETP mRNA in both liver and spleen, about 4.5- and 13-fold, respectively. The E9-deleted CETP mRNA did not increase in response to the high cholesterol diet or the apoE0 background, and increased less than the FL CETP mRNA in the compound mice on the high cholesterol diet. Thus, the ratio of FL/E9-deleted mRNA was significantly increased in liver and spleen in NFR.CETP(±E9) Tg mice on the high cholesterol diet (Table 3), and was increased further in the compound mice on the high cholesterol diet. Thus, the induction of CETP gene expression by hypercholesterolemia favors the FL form of CETP mRNA over the E9-deleted form. Paralleling the mRNA changes, plasma CETP activity was increased from 5.8 ± 3% in NFR.CETP(±E9) Tg mice on chow to 9.5 ± 3% on the high cholesterol diet to 59 ± 9% in NFR.CETP(±E9) Tg/apoE0 mice on the high cholesterol diet.
Next we studied CETP mRNA expression in NFR.CETP(±E9) Tg mice during development (Fig. 7). Hepatic expression of full-length CETP mRNA increased at the end of embryonic development, peaked in suckling mice, and decreased in adults. In the small intestine, there was substantial expression of the CETP mRNA at birth (day 0), but expression was extinguished in adults. In contrast to these findings, in the spleen CETP mRNA was not detected in fetal mice, was low in the neonatal period, and increased to highest levels in adult animals.
Both full-length and E9-deleted forms were present at all times that the CETP mRNA was detected, but there were subtle differences in the pattern of expression. The developmental changes were quantitated by PhosphorImager and are depicted in Fig. 8; the data are averaged for three separate developmental studies in pools of three to four mice/study. In the liver, the peak of both FL and E9-deleted CETP mRNAs was seen in day 7 suckling mice. Prior to this the full-length form increased while the E9-deleted form did not, so that the highest ratio of full-length to E9-deleted mRNA (2.2) occurred at birth. In the spleen, there was a sharp increase in CETP mRNA between day 7 and 17 mice, associated with an increase in the ratio of full- length/E9-deleted mRNA, and highest levels of FL mRNA were observed at day 60. Thus, in the liver and spleen there was an increase in the ratio of FL/E9-deleted mRNA prior to the peak of FL mRNA expression. In the small intestine, both forms were expressed in neonatal mice and extinguished following the suckling period. Expression levels after day 0 were too low to allow accurate quantitation of ratios of the two mRNA forms.
Figure 8: Developmental changes in CETP mRNA in liver (A), spleen (B), and small intestine (C), showing changes in FL and exon 9- deleted mRNA forms and in the ratio of FL/exon 9deleted forms. The data shown are the mean results from two or three different studies, each involving pools of three to four mice per time point. CETP RNA was determined by RNase protection assay of total RNA (100 µg) and quantitated by PhosphorImager; RNA recovery was corrected to an internal standard (28 S rRNA).
In general, alternative splicing of genes either serves as a regulatory device to modulate levels of gene expression or as a way to produce variant protein isoforms with differing functions. The present findings suggest that alternative splicing of the CETP gene falls into the first category and support the hypothesis that omission of E9 sequences represents a mechanism to limit the formation of active plasma CETP. Mice expressing a CETP transgene with E9 sequences omitted had no detectable plasma lipid transfer activity and no changes in plasma lipoprotein profile, showing that E9-deleted sequences are inactive. Furthermore, studies in mice expressing a CETP transgene with optional splicing of exon 9 indicated that inclusion of E9 was favored under circumstances where CETP mRNA was increased.
Studies of mice
containing the mT-CETP transgene with E9 sequences omitted
(mT.CETP(-E9) Tg mice) suggested that expression of E9-deleted
transcripts was curtailed on several levels. Following zinc induction,
there was massive expression of E9-deleted CETP mRNA to levels more
than 100-fold that of the FL CETP mRNA in the NFR.CETP(+E9) Tg
mice (Fig. 2). However, compared to the FL CETP protein, there
was no corresponding overexpression of E9-deleted protein in cell
homogenates or in plasma ( Fig. 3and Fig. 4). Most
likely, the E9-deleted mRNA is poorly translated. An alternative
explanation, i.e. that the E9-deleted protein is unstable,
seems less likely, since pulse-chase experiments in cell lines stably
expressing both forms of CETP show that the intracellular half-life of
the E9-deleted protein is much longer than the full-length form. ()As in the cell culture studies (Quinet et al.,
1993; Inazu et al., 1992), the E9-deleted protein appeared to
be relatively poorly secreted, since it was more abundant than the FL
form in cell homogenates but less abundant in plasma (cf. Fig. 3and Fig. 4). The E9-deleted form of CETP has not
been detected in human plasma (Inazu et al., 1992); its
presence in the mT.CETP(-E9) Tg mice probably results from
overexpression. Finally, even though these mice had some E9-deleted
protein in plasma, it bound to HDL poorly and was inactive in lipid
transfer. Site-directed mutagenesis studies show that two positively
charged amino acids contained within E9 are essential for HDL binding
and optimal lipid transfer activity (Jiang et al., 1995).
One aspect of the cell culture studies which was apparently not confirmed in the Tg mice was the dominant inhibition of the secretion of full-length active CETP by the E9-deleted protein (Quinet et al., 1993). Perhaps in the cell transfection studies full-length and E9-deleted proteins were more highly concentrated in the endoplasmic reticulum than in vivo, leading to formation of poorly secreted hetero-dimers (Quinet et al., 1993). The negative studies using compound mT.CETP (-E9)/NFR.CETP(+E9) mice do not completely exclude this as potential regulatory mechanism, because it is possible that the temporal and spatial expression of the two forms of CETP mRNA was different in the compound mice, since the two mRNAs were being formed off separate promoters. In this regard, the 10-fold increase in plasma CETP activity induced by hypercholesterolemia in the NFR.CETP(±E9) mice exceeded the 4.5-fold increase in hepatic CETP mRNA, whereas in NFR.CETP(+E9) mice changes in plasma CETP closely parallel CETP mRNA changes (Masucci-Magoulas et al., 1996). Thus, the alternative splicing mechanism may have contributed to the induction of plasma CETP activity in NFR.CETP(±E9) mice.
Regulatory studies in NFR.CETP±E9 transgenic mice showed an increased ratio of FL/E9-deleted CETP mRNA when CETP gene expression was increased in response to hypercholesterolemia and during development. In the developmental studies the increase in ratio of hepatic FL/E9-deleted mRNA preceded the peak of mRNA expression, suggesting that changes in splicing may occur more rapidly than overall changes in mRNA abundance. The results of the studies in NFR.CETP±E9 Tg mice are consistent with a regulatory role of alternative splicing. However, the changes in the splicing pattern were moderate and the largest change in ratio of FL/E9-deleted mRNA was only about 2-fold. However, the change in ratio of FL/E9-deleted mRNA was the most sensitive index of diet response (Table 3). Thus, alternative splicing may be a subtle mechanism to control the amount of active CETP that is formed, perhaps with different kinetics to transcriptional or other control mechanisms, as suggested in the developmental studies (Fig. 8). Our studies do not differentiate whether altered ratios of full-length/E9-deleted mRNA arise from regulation of splicing per se, or altered stability of the different mRNA forms.
A major novel finding of the present
study was that CETP gene expression was found to be markedly influenced
by developmental changes. In the line of mice expressing the
NFR.CETP(±E9) transgene, hepatic and intestinal expression
peaked in suckling or newborn mice, while splenic expression was
highest in adults. Although these findings will need to be confirmed in
a species that naturally expresses CETP, they imply strong
developmental control of CETP gene expression. The intestinal
expression could indicate a role in cholesterol absorption or
chylomicron metabolism in the newborn animal. Hepatic expression may
coincide with the induction of enzymes of lipid biosynthesis and
increased secretion of VLDL. The different time course of expression in
the spleen suggests a distinctive role. In hamsters and humans the CETP
mRNA appears to be expressed primarily in lymphocytes; in humans
splenic lymphocytes contain about equal amounts of FL and E9-deleted
mRNA (Jiang et al., 1991). ()Thus, the increase in
expression in the spleen could parallel maturation of the immune
system. Implicit in this discussion of tissue-specific functions during
development is the idea that CETP must somehow be locally concentrated
and active within organs, analagous to apoE (Mahley, 1988).
Recent studies show decreased atherosclerosis in hypertriglyceridemic CETP transgenic mice (Hayek et al., 1995) and increased coronary heart disease in humans with genetic CETP deficiency (Zhong et al., 1995). Thus, increasing CETP expression in humans could be an antiatherogenic treatment. Marked changes in the formation of active plasma CETP, reflecting coupled changes in transcription and splicing, suggest that modulation of CETP gene expression could be a therapeutic option.