Increased Production of Apo B-Containing Lipoproteins in the Absence of

Hyperlipidemia in Transgenic Mice Expressing Cholesterol-7 -hydroxylase*

Jon H. Miyake†, Xuan Dao T. Doung

†, William Strauss

†, Gina L. Moore

†,

Lawrence W. Castellani‡, Linda K. Curtiss

§, John M. Taylor

¶ and Roger A. Davis

γ

†Mammalian Cell and Molecular Biology Laboratory,

San Diego State University, San Diego, CA 92182-4614

‡Department of Microbiology and Molecular Genetics, UCLA, Los Angeles, CA

§The Scripps Research Institute, La Jolla, CA

¶Gladstone Institute for Cardiovascular Research, University of California, San Francisco,

San Francisco, CA

γAddress correspondence to:

Roger A. DavisMammalian Cell and Molecular Biology LaboratoryLife Sciences Building LS3075500 Campanile DriveSan Diego State UniversitySan Diego, CA 92182-4614

(619) 594-7936 Telephone(619) 594-7937 FaxE-Mail: rdavis@sunstroke.sdsu.edu

Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on April 25, 2001 as Manuscript M101853200 by guest on A

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RUNNING TITLE: Induction of VLDL Production by CYP7A1

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Summary

The finding that expression of a cholesterol-7α-hydroxylase (CYP7A1) transgene in

cultured rat hepatoma cells caused a coordinate increase in lipogenesis and secretion of

apolipoprotein B (apo B)-containing lipoproteins led to the hypothesis that hepatic

production of apo B-containing lipoproteins may be linked to the expression of CYP7A1

(Wang et al, J. Biol. Chem. 272:19351-19358 (1997). To examine this hypothesis in vivo,

a transgene encoding CYP7A1 driven by the constitutive liver-specific enhancer of the

human apo E gene was expressed in C56BL/6 mice. The expression of CYP7A1 mRNA

(20-fold), protein (~10-fold) and enzyme activity (5-fold) was increased in transgenic mice,

compared to non-transgenic littermates. The bile acid pool of CYP7A1 transgenic mice was

doubled mainly due to increased hydrophobic di-hydroxy bile acids,

taurochenodeoxycholate (10-fold) and taurohyodeoxycholic acid (5-fold); whereas the more

hydrophilic bile acids, tauromuricholic acid (-50%) and taurocholic acid (-90%), were

decreased. In CYP7A1 transgenic mice, livers contained ~3-fold more SREBP2 mRNA

and no significant increase in SREBP1 mRNA. Hepatic expression of mRNAs encoding

lipogenic enzymes (i.e., fatty acid synthase, acetyl-CoA carboxylase, stearoyl CoA

desaturase, squalene synthase, farnesyl pyrophosphate synthase, HMG-CoA reductase and

low density lipoprotein (LDL) receptor) as well as microsomal triglyceride transfer protein

were elevated ~3 to 5-fold in transgenic mice. CYP7A1 transgenic mice also displayed > 2-

fold increase in hepatic production and secretion of apo B-containing lipoproteins. In spite

of the increased hepatic secretion of apo B-containing lipoproteins in CYP7A1 mice,

lipoprotein levels in plasma did not increased. These data suggest that the 5-fold increased

expression of the LDL receptor displayed by the livers of CYP7A1 transgenic mice was

sufficient to compensate for the 2-fold increase production of apo B-containing

lipoproteins. These findings emphasize the important homeostatic role that CYP7A1 plays

in balancing the anabolic lipoprotein assembly/secretion pathway with the cholesterol

catabolic bile acid synthetic pathway.

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Introduction

Hepatic lipoprotein secretion requires: apolipoprotein B (apo B)1 having a size that

is sufficiently large to allow the formation a lipoprotein particle containing a neutral lipid

core; the availability of lipids (i.e. phospholipids, triglycerides, cholesterol and cholesterol

esters) and the intralumenal chaperone/lipid transfer protein microsomal triglyceride transfer

protein (MTP) (reviewed in 1-6). The assembly of apo B-containing lipoproteins is

abrogated when these essential requirements are not satisfied, resulting in rapid degradation

of apo B within the hepatocyte (7). The most characterized pathway responsible for the

rapid, co-translational degradation of incompletely translocated apo B is via an ubiquitin-

dependent proteasome process (8-11). Several additional pathways that may contribute to

the intracellular degradation of apo B have been described (12-15).

Cholesterol-7α-hydroxylase (CYP7A1) is a liver-specific enzyme that regulates the

production of bile acids from cholesterol (16-18). Previous studies using cultured rat

hepatoma cells showed that stable expression of CYP7A1 increased the cellular content of

mature sterol response element binding protein 1 (SREBP1) as well as mRNAs encoding

essentially all the lipogenic enzymes required for very low density lipoprotein (VLDL) lipid

production and assembly and secretion of apo B100-containing lipoproteins (19). These

findings led us to hypothesize that CYP7A1 expression may indirectly regulate the

assembly and secretion of VLDL via increasing the expression of SREBP, the expression

of lipogenic enzymes and the expression of MTP (4,11,19,20).

The coordinate induction of VLDL assembly and secretion observed in rat hepatoma

cells expressing a CYP7A1 transgene was reminiscent of the coordinate changes in the

biosynthesis of VLDL lipids and secretion of apo B observed in primary hepatocytes

obtained from carbohydrate-induced (21) and fasted rats (22-24). Subsequent studies of

livers from fasted and re-fed carbohydrate induced mice showed that changes in the hepatic

content of SREBPs could account for the observed changes in fat and sterol metabolism

(25). Fasted mice displayed reduced levels of SREBP1c and SREBP2, while livers from

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carbohydrate re-fed mice displayed mainly an increase in SREBP1c (SREBP2 returned to

normal levels) (25). SREBP1c preferentially increases the transcription of mRNAs

involved in fatty acid biosynthesis (26-28). In contrast, SREBP2 appears preferentially

increase the transcription of mRNAs involved in cholesterol metabolism (26). Thus the

relative balance between mature levels of SREBP1 compared to SREBP2 influences

metabolism via changing the expression of their gene targets.

In the studies reported here, we examine the effect of expressing a CYP7A1

transgene in mice on the hepatic expression of mRNAs encoding SREBP1 and SREBP2,

lipogenic enzymes, the low density lipoprotein (LDL) receptor and MTP and relate these

changes to the production of hepatic apo B-containing lipoproteins. The results

demonstrate that in CYP7A1 transgenic mice, hepatic production of apo B-containing

lipoproteins is significantly augmented; yet there is no accumulation in plasma. Thus,

plasma levels of lipoproteins do not necessarily reflect relative rates of VLDL production by

the liver.

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Methods

Transgenic vector

Transgenic mice expressing the rat CYP7A1 cDNA were generated at the Gladstone

Institute of Cardiovascular Disease, University of California, San Francisco, San Francisco,

CA. The transgene (pLiv.7 plasmid) was constructed using a DNA construct containing the

hepatic control region of the human apolipoprotein E3 gene ((29) a gift from John M.

Taylor). The rat CYP7A cDNA (1.8 kb) which contained all the coding region and 58 bp of

3' untranslated region with EcoRI sites on both ends was ligated into the pLiv plasmid at the

MfeI site (29). When expressed in cultured cells, this vector produced a single CYP7A

mRNA (2.4) species that is distinct from the endogenous mouse CYP7A1 mRNA multiple

bands around 4.2 kb. After establishing the correct orientation of the cDNA the transgenic

vector was isolated from bacterial sequence by using SacII and SpeI restriction enzymes.

The 7.6kb transgenic construct was gel purified using a QIAquick gel extraction kit

(Qiagen). The construct was microinjected into single cell embryos of C57BL/6J X SJ

strain and implanted into pseudo-pregnant female mice.

CYP7A1 transgenic mice

Tails of pups were clipped, DNA obtained by DNAEasy Qiagen kit and PCR

performed using the appropriate vector specific primers. Pups (fo) showing genomic

integration of the desired transgenic sequences were bred with C57BL/6J mice producing

two founder lines of f1 mice that expressed rat CYP7A1 mRNA produced from the

transgene. Male and female progeny from these two lines of CYP7A1 transgenic mice were

observed to express similar levels of rat CYP7A1 mRNA. In order to obtain a line of

CYP7A1 transgenic mice having a C57BL/6J background, male mice produced from each

subsequent mating were bred with female C57BL/6J mice (Jackson Labs). The

experimental results reported herein were obtained from CYP7A1 transgenic mice and their

non-transgenic littermates that were obtained from matings of male progeny produced from

a total of 5 back crosses with female C57BL/6J mice. We estimate that the genetic

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background of the transgenic and non-transgenic mice is >90% C57BL/6J. The transgenic

mice were morphologically indistinguishable from normal C57BL/6 mice. They displayed

normal bodyweight, fecundity, litter size, fecal consistency and appetite. Mice were housed

in a room having a 12 hour light (6 am-6 pm) and 12 hour dark (6 pm-6 am) cycle. The

mice were fed Purina breeder chow and drinking water ad libitum.

Plasma lipids

Mice were anesthetized with isofluorane. Blood was collected by retro-orbital

puncture into heparanized Natelson tubes. Aliquots of plasma were subjected to lipid

(cholesterol, cholesterol ester, triglyceride and phospholipid) analysis using commercially

available enzymatic kits and calibration standards (Sigma), as described (30). Cholesterol

concentration in apo B lipoproteins was derived by subtracting the concentration of plasma

high density lipoprotein (HDL) cholesterol from that of plasma total cholesterol. For fast

performance liquid chromatography (FPLC) analysis, 450 µl of pooled heparinized plasma

was injected onto two Pharmacia (Uppsala, Sweden) Superose 6 columns connected in

series, and lipoproteins eluted with 154 mM NaCl and 3 mM sodium azide in endotoxin-

free water, pH 8.2. Fractions of 1.0 ml each were collected. The lipoprotein elution profile

was determined by measuring cholesterol.

CYP7A1 RNase Protection Assay

In vitro transcribed radiolabeled antisense and sense strand RNAs were synthesized

according to manufacturer’s protocol using T7 RNA polymerase (Maxiscript, Ambion).

The resulting transcripts were gel purified using a 5% denaturing acrylamide gel. The

transcripts were excised and eluted from the gel matrix and subsequently ethanol

precipitated. Approximately 30,000 cpm of the in vitro transcript were used to hybridize to

20 ug of total RNA. The RNase protection assay was performed using a kit (HybSpeed,

Ambion) with RNase T1 and RNaseA, following manufacturer specifications. The

protected RNA fragments were run through a 5% denaturing acrylamide gel and visualized

by PhosphorImager analysis (Molecular Dynamics).

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Northern Analysis .

Mice were anesthetized with isofluorane and exsanguinated. Livers were excised

and immediately frozen in liquid nitrogen. The frozen tissue was homogenized with a

Tekmar tissue homogenizer in 5 ml 4M guanidinium thiocyanate and total RNA processed

as previously described (31). Poly (A) RNA was isolated and electrophoresed through a

0.8% agarose formaldehyde gel. The gel was blotted by capillary action onto nitrocellulose

(Zetaprobe, Biorad) and the RNA fixed by ultraviolet crosslinking (Stratalinker, Stratagene).

The blot was then pre-hybridized and hybridized with 1-5 X 106 cpm/ml

32P nick translated

cDNA probes prepared from gel-purified inserts for the indicated genes at 440

C in roller

bottles in a hybridization oven (Labline) in 0.12M NaPO4, 0.25M NaCl, 7% SDS, 50%

deionized formamide. The blot was washed sequentially in 2X SSC, 0.1% SDS; 0.5X SSC,

0.1% SDS and 0.1X SSC, 0.1% SDS for 30 min each at 440

C. Blots were stripped of the

probe and re-hybridized sequentially with the indicated cDNA probes.

CYP7A1 enzyme activity

Mouse liver microsomes were isolated and the enzyme activity of CYP7A1 was

determined by high pressure liquid chromatography (HPLC) using [414C] cholesterol

(30,32).

Hepatic Triglyceride production

Triton WR-1339 (Sigma) solution (15g/dl in 0.9% NaCl) was injected at 5mg/kg

body weight via tail vein after an overnight (15 hour) fast. Blood was collected retro-

orbitally every hour up to 4 hours post injection. Under these conditions plasma VLDL

clearance is inhibited. Plasma triglycerides were assayed as previously described (30).

De novo synthesized [35

S]methionine-labeled apo B

Mice were injected with 1 mCi of [35

S] labeling mix (NEN, NEG-772 Express-

[35

S] Protein Labeling Mix) intra-peritonealy after an overnight fast. After 2 hours the mice

were injected with Triton WR-1339 (Tyloxapol, Sigma) by tail vein. Blood was collected

every hour post injection for 4 hours. Plasma proteins were run through a 2-15% SDS-

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PAGE. The gel was dried and exposed to autoradiography. Apo B100 and apo B48

proteins were separately excised from the gel and solubilized in 2 ml 90% Hyamine

Hydroxide (ICN) at 450

C for 24 hours in scintillation vials. 10 ml of Cytoscint (ICN) was

added and the radioactivity assayed by a β-scintillation (Beckman Inc.).

Western Blot Analysis.

Western blotting was performed as described (33). Briefly, 30 µg of microsomes or

1:50 dilution of plasma of each sample was electrophoresed by SDS-PAGE (5-15%

gradient), the gels were then electroblotted onto nitrocellulose (Schleicher and Schuell). The

non-specific binding sites of the membranes were blocked using 10% defatted dried milk,

followed by addition of the indicated primary antibody. The relative amount of primary

antibody bound to the proteins was detected with the species-specific horseradish

peroxidase-conjugated IgG. After washing, blots were developed using the ECL detection

kit (Amersham) on high performance chemiluminescence film (Hyper film ECL,

Amersham). Polyclonal antibodies were used against apo B, MTP, CYP7A1 and protein

disulfide isomerase (PDI).

Bile Acid Pool Analysis

The bile acid pool size was quantitated by HPLC (34). Upon sacrifice under

anesthesia, the abdomen was opened and the gallbladder, liver, and small intestine were

excised and homogenized in 5 ml of 100 % ethanol. [2414

C] taurocholate was added to the

homogenate as a recovery standard. The homogenate was centrifuged at 1,700 rpm for 20

minutes. The supernatant was concentrated under a nitrogen stream and resuspended in

distilled water. This was run through C18 PrepSep Column and eluted with 100 %

methanol. The sample was concentrated to 500 µl volume and 10 µl of bile acid sample

loaded onto having a Beckman ultrasphere C18 HPLC column under isocratic elution using

67% methanol:water containing 0.01M KH2PO4, pH 5.4. The flow rate was 0.75

ml/minute and absorbance was read at 205 nm. Bile acid size pool was calculated using

known standards and the [2414

C]-taurocholate recovery standard.

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Statistical analysis

Results are given as mean ± standard deviation (S.D.). Statistical significance was

determined by Student’s t test using double-tailed p values. Values of p < 0.05 were

considered to be significant.

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Results

We constructed a transgenic vector that we anticipated would provide a stable, high

level of expression of CYP7A1 by the liver. A construct containing the coding region of rat

CYP7A1 and the liver-specific enhancer region of the human apo E gene promoter region

(29) was found to express high levels of CYP7A1 mRNA when transiently transfected into

HepG2 cells (data not shown). This transgene was subsequently injected into blastocysts,

which were implanted into pseudo pregnant female mice (29). Approximately 30% of the

newborn mice were shown to contain the rat CYP7A1 transgene, as determined by PCR

analysis of genomic DNA obtained from the tail.

First generation transgenic mice were bred with C57BL/6J mice. Progeny from

each mating were examined for the presence of rat CYP7A1 mRNA (derived from the

transgene) in liver. Two litters expressed rat CYP7A1 at levels similar to those reported in

Figure 1. There was no significant difference in the expression of rat CYP7A1 mRNA

between male and female mice. These transgenic mice also displayed increases in the

hepatic expression of the LDL receptor mRNA and SREBP2 mRNA that were similar to

those displayed by fifth generation transgenic mice having a genetic background that is

estimated to be >90% C57BL/6J (Figure 3). Thus, the phenotype described below for

CYP7A1 transgenic mice is not likely the result of an epigenetic event caused by integration

of the transgene.

CYP7A1 transgenic mice express CYP7A1 mRNA, protein and enzyme activity at

constitutively high levels

Rat (derived from the transgene) CYP7A mRNA was clearly evident in the livers of

CYP7A1 transgenic mice, whereas it was undetected in the livers obtained from non-

transgenic mice (Figure 1A). The mRNA expression level of the rat CYP7A1 transgene

was estimated to be >50-fold greater than the endogenous mouse CYP7A1. The expression

of the transgene mRNA appeared to be liver-specific (i.e. using the RNase protection assay,

we could not detect rat CYP7A1 mRNA in brain, lungs, heart, kidneys and skeletal muscle-

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data not shown). Western blots of microsomes obtained from the 3 livers of transgenic

mice displayed >9-fold more immunoreactivity toward an immunoaffinity purified antibody

made against rat CYP7A1 compared to microsomes from non-transgenic mice (Figure 1B).

The increased expression of CYP7A1 mRNA and protein in transgenic mice resulted in a

~6-fold increase in CYP7A1 enzyme activity of hepatic microsomes (Figure 1C).

Transgenic expression of CYP7A1 increases the endogenous bile acid pool due mainly

to increased taurochenodeoxycholate

The bile acid pool was about 2-fold greater in CYP7A1 transgenic mice compared to

their non-transgenic littermates (Figure 2A). It is interesting to note the stark difference in

biliary bile acid composition between the CYP7A1 transgenic mice and non-transgenic

mice. In the CYP7A1 transgenic mice, the relative content of the more hydrophobic

(dihydroxy) biliary bile acids, taurochenodeoxycholic acid (+10-fold) and

taurohyodeoxycholic acid (+5-fold), were increased, whereas the relative content of the

more hydrophilic (trihydroxy) bile acids, taurocholic acid (-90%) and tauromuricholic acid

(-50%), were significantly decreased (Figure 2B).

Transgenic expression of CYP7A1 increases the hepatic expression of lipogenic genes

Compared to livers of non-transgenic littermates, livers of CYP7A1 transgenic mice

displayed significantly greater levels of mRNAs encoding enzymes involved in fatty acid

synthesis (acetyl-CoA carboxylase: 4.3-fold increase and fatty acid synthase: 5.8-fold

increase) and cholesterol metabolism (HMG-CoA reductase: 5.9-fold increase, farnesyl

diphosphate synthase: 3.9-fold increase, squalene synthase: 4.9-fold increase and the LDL

receptor: 5.2-fold increase) (Figure 3). While the level of SREBP1 mRNA was similar in

both groups of mice, in CYP7A1 transgenic mice, SREBP2 mRNA levels were 3-fold

greater than non-transgenic littermates (Figure 3).

The livers of CYP7A1 transgenic mice also displayed increased expression of

mRNAs encoding MTP (3-fold increase) and stearoyl CoA desaturase (SCD-1) (4-7-fold

increase), two gene products thought to be required for the assembly and secretion of apo

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B-containing lipoproteins (35,36) (Figure 3). Consistent with previous studies indicating

that hepatic apo B mRNA expression is resistant to changes in expression (24), the livers of

both groups of mice showed similar levels of apo B mRNA (Figure 3). It is interesting to

note that the changes in the expression of mRNAs displayed by CYP7A1 transgenic mice is

similar to those observed in SREBP-2 transgenic mice (26). Increased expression of

hepatic LDL receptors was also observed in hamsters expressing CYP7A1 via an

adenovirus transgene (37).

In CYP7A1 transgenic mice, the expression of endogenous (mouse) CYP7A1 was

reduced to undetectable levels (Figure 3), a result expected from the doubling of the

endogenous bile acid pool size (Figure 2). The alternative (acidic) bile acid biosynthetic

pathway is controlled by oxysterol-7α-hydroxylase (CYP7B1) mRNA (38,39). The livers

of CYP7A1 transgenic mice expressed a -4-8-fold reduction in the expression of CYP7B1

compared to the livers of their non-transgenic siblings (Figure 3). Feeding mice bile acids

has been reported to cause a modest reduction in the hepatic expression of CYP7B1 mRNA

(39).

Transgenic expression of CYP7A1 increases MTP protein levels

To further investigate MTP expression in the CYP7A1 transgenic mice we

performed western blot analysis of hepatic microsomes from transgenic and non-transgenic

mice at mid-light. MTP protein levels are increased ~1.7 fold, p<0.025, in CYP7A1

transgenic mice compared to non-transgenic littermates when normalized to Protein

Disulfide Isomerase (PDI) (data not shown).

Transgenic expression of CYP7A1 increases the assembly and secretion of triglyceride-

rich lipoproteins

We quantitated the relative rate of accumulation of triglycerides and [35

S]

methionine-labeled apo B in the blood of mice treated with Triton WR-1339. Triton WR-

1339 prevents the metabolism and removal of lipoproteins from plasma (40). The rate of

accumulation of VLDL in plasma following intravenous injection of Triton WR-1339

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therefore provides a means to estimate their production rates. Following Triton WR-1339

administration, there were significantly greater amounts of triglyceride that accumulated in

the plasma of CYP7A1 transgenic mice compared to non-transgenic littermates (Figure 4).

Furthermore, least squares analysis of the linear rate of accumulation of plasma triglycerides

showed that CYP7A1 transgenic mice displayed a slope that was 2.2-fold greater than that

obtained from non-transgenic mice.

The rates of accumulation of [35

S]methionine-labeled apo B100 (1.65-fold,

p<0.025) and apo B48 (+24%, p<0.05) as determined by the slopes of the linear

accumulation of [35

S]methionine-labeled apo B versus time in h were also increased in

CYP7A1 transgenic mice compared to non-transgenic littermates (Figure 5). These

combined data indicate that the hepatic assembly and secretion of apo B-containing

lipoproteins is increased in CYP7A1 transgenic mice.

In spite of increased hepatic production of apo B100-containing lipoproteins, CYP7A1

mice display no increase in plasma or hepatic lipids

In contrast to the significant increased production of hepatic apo B-containing

lipoproteins displayed by CYP7A1 transgenic mice, lipoproteins did not accumulate in

plasma (Figures 6A-6D). While the concentration of triglyceride was slightly increased in

the plasma of CYP7A1 mice, this 12% increase was not statistically significant.

Furthermore, the plasma levels of cholesterol in non-HDL lipoproteins were significantly

decreased in CYP7A1 transgenic mice (Figure 6A, -50%, p<0.01). We also examined the

cholesterol content of lipoproteins that were fractionated by FPLC. The results confirm the

previous results showing that in the plasma of CYP7A1 transgenic mice the cholesterol

content of the apo B-containing lipoproteins (intermediate density lipoprotein (IDL) and

LDL, fractions 21-36; Figure 6C) contained significantly less cholesterol. Plasma from

CYP7A1 transgenic mice also contained significantly less cholesterol in the HDL fractions

(fractions 36-51-Figure 6C). While the plasma of CYP7A1 transgenic mice contained less

cholesterol compared to the plasma obtained from non-transgenic littermates, the plasma

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content of both apo B100 and apo B48 was similar (Figure 6D). Thus, in spite of the

significant 2-fold increase in hepatic production of apo B-containing lipoproteins in

CYP7A1 mice, lipoproteins did not accumulate in plasma.

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Discussion

Our results show that hepatic "over-expression" of a CYP7A1 transgene in mice

leads to a 2-fold increase in the production of apo B100-containing lipoproteins. Further

analysis indicates that the increased hepatic lipoprotein assembly/secretion displayed by

CYP7A1 mice occurs in response to an induction of lipogenic biosynthetic enzymes whose

transcription is increased by SREBP. The associated induction by SREBP2 of hepatic

expression of LDL receptor mRNA was sufficient to prevent the accumulation in plasma of

apo B-containing lipoproteins in spite of the increased lipoprotein production displayed

CYP7A1 transgenic mice. The apparent coordinate linkage of the cholesterol/bile acid

catabolic pathway with the anabolic lipoprotein assembly pathway contributes to the

maintenance of cholesterol and lipoprotein homeostasis in C57BL/6 mice.

The CYP7A1 transgene provided constitutive high level expression of CYP7A1

mRNA, protein and enzyme activity in the livers of recipient mice (Figure 1). Since

transgenic mice showed normal fecundity, pregnancy, size of the litters, sex distribution,

weight gain, fecal consistency, general health and longevity (data not shown), the artificially

increased expression of CYP7A1 did not impair essential physiologic functions. It is

interesting to note that the size of the bile acid pool of CYP7A1 transgenic mice was only

increased ~2-fold (Figure 2), whereas CYP7A1 enzyme activity increased ~6-fold (Figure

1C). Thus, the expansion of the bile acid pool size was disproportionately less than

expected. In other studies of mice fed a fed diet containing 0.2% cholate, the bile acid pool

was also increased only 2-fold (34). These combined data suggest that an as yet to be

defined process may limit expanding the bile acid pool of mice to beyond 2-fold.

The finding that the expression of CYP7B was decreased ~70% (Figure 3) suggests

that bile acid production by the alternative bile acid synthetic pathway (38,41) was

diminished in CYP7A1 transgenic mice. Thus, in CYP7A1 transgenic mice, the alternative

bile acid synthetic pathway contributes less to the bile acid pool whereas the CYP7A1

dependent pathway contributes more. It has been generally noted that dihydroxy-bile acids

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(e.g., chenodeoxycholic acid) are the preferential products of the alternative (oxysterol-

derived) bile acid synthetic pathway (17). It is therefore somewhat surprising that the bile

acid pool of CYP7A1 transgenic mice contains relatively more hydrophobic dihydroxy-bile

acids and less hydrophilic tri-hydroxy bile acids (e.g., increased taurochenodeoxycholic

acid and less taurocholic and tauro-β-muricholic acids-Figure 2B). The marked decrease in

cholic acid may be explained by bile acid repression of CYP8B1 which diverts de novo

synthesized intermediates from forming chenodeoxycholic acid so that they produce cholic

acid (42,43). Thus, the doubling of the bile acid pool in CYP7A1 transgenic mice may have

repressed 12α-hydroxylation causing a compensatory increase in taurochenodeoxycholic

acid.

The recent discovery demonstrating the importance of bile acid structure on

activating the ligand-dependent farnesoid-X-receptor transcription factor, which regulates

the expression of CYP7A1, the ileal bile acid binding protein (44-46) and the canalicular

bile acid export protein (47), emphasizes the complex inter-relationships between bile acid

pool composition, gene expression and physiology of bile acids. CYP7A1 transgenic mice

may provide an experimental model to explore the mechanisms that determine the

composition of the bile acid pool.

A major impetus for undertaking these studies was to examine the influence that

CYP7A1 has on hepatic production of apo B-containing lipoproteins. The results of these

studies show for the first time that augmented expression of CYP7A1 via transgenic

constitutive expression in mice increases the production of apo B-containing lipoproteins by

increasing the hepatic expression of mRNAs whose transcription is increased by mature

SREBP (Figure 3). These mRNAs include lipogenic enzymes regulating the synthesis of

fatty acids (e.g., acetyl-CoA carboxylase (48), fatty acid synthase (49) and SCD-1 (36)) and

cholesterol (e.g., HMG-CoA reductase (50), farnesyl diphosphate synthase (51) and

squalene synthase (52)). There were also increased hepatic expression of MTP and

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SREBP2 mRNAs in CYP7A1 transgenic mice, whereas the expression of apo B mRNA

was similar in both groups (Figure 3).

These changes in the expression of lipogenic enzymes were associated with

increased production of apo B-containing lipoproteins (Figures 4 and 5). In the mouse apo

B100 is exclusively derived from the liver (53-55). Thus, the concordant 2-fold increase in

the accumulation of both triglycerides (Figure 4) and [35

S] methionine-labeled apo B100

(Figure 5) displayed by CYP7A1 transgenic mice treated with Triton WR-1339 suggests

that hepatic VLDL assembly and secretion was coordinately increased by CYP7A1.

Lipogenesis is a major determinant of how much apo B enters the VLDL

assembly/secretion pathway and how much is degraded by the alternative ubiquitin-

dependent proteasome pathway (reviewed in (4)). The increased production of apo B100-

containing lipoproteins without a change in apo B mRNA expression displayed by CYP7A1

transgenic mice is consistent with this proposal.

The increased production of apo B-containing lipoproteins displayed by CYP7A1

transgenic mice is similar to the phenotype displayed by rat hepatoma cells (McA-RH7777

cells) that expressed a CYP7A1 transgene (19). There is one potentially important

difference. In the livers of CYP7A1 transgenic mice SREBP2 mRNA was selectively

increased (Figure 3), whereas in rat hepatoma cells (McA-RH7777 cells) expressing a

CYP7A1 transgene SREBP1 was selectively increased (19). Our findings showing that

CYP7A1 transgenic mice displayed increased expression of SREBP2 mRNA are consistent

with those showing that SREBP2 is induced in livers of hamsters treated with a regimen that

decreases cellular cholesterol levels (i.e. a HMG-CoA reductase inhibitor and a bile acid

binding resin) (56). The basis for why CYP7A1 expression in rat hepatoma cells (McA-

RH7777 cells) increases the expression of SREBP1, but not SREBP2 (19) may be related

to cellular differences in oxysterol metabolism. Recent studies show that treating rat

hepatoma cells (McA-RH7777 cells) with a HMG-CoA reductase inhibitor selectively

increases SREBP1c due to an oxysterol-mediated activation of LXR (28).

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The additional finding that MTP mRNA was increased in the livers of SREBP2

transgenic mice (26) is consistent with the proposal that the increase in SREBP2 mRNA in

the livers of CYP7A1 transgenic mice may mediate the increased MTP mRNA expression.

Several studies support the concept that in mice MTP expression may be rate-limiting for

the production of apo B-containing lipoproteins (57-60). The increased hepatic expression

of MTP mRNA together with the increased expression of lipogenic enzymes contributes to

the increased production of apo B-containing lipoproteins displayed by CYP7A1 transgenic

mice.

In spite of the increased production of apo B-containing lipoproteins displayed by

CYP7A1 transgenic mice treated with Triton WR-1339, the plasma from these mice showed

no evidence of increased lipoproteins (Figure 6). These data suggest that the capacity for

metabolizing and removing apo B-containing lipoproteins from plasma of CYP7A1

transgenic mice exceeded the 2-fold increase in their production. These metabolic

processes include lipoprotein lipase and hepatic lipase mediated lipolysis followed by

removal from plasma of the resulting remnant particles by the LDL receptor (61-66).

SREBP2 is a more potent inducer of the LDL receptor, than is SREBP1 (26,28).

LPL expression can be also activated by SREBP-2 (67). Thus, the selective increased

expression of SREBP2 mRNA in the livers of CYP7A1 transgenic mice is likely to be

responsible for the 5.2-fold increased expression of LDL receptor mRNA (Figure 3).

Increased hepatic expression of LDL receptors may also account for the significant

reduction of IDL, LDL cholesterol in the plasma of CYP7A1 transgenic mice (Figure 6).

Our findings are consistent with those showing that the livers of hamsters expressing a

CYP7A1 adenovirus transgene display increased expression of the LDL receptor, increased

rates of hepatic LDL clearance and reduced plasma LDL cholesterol (37).

In humans, some moderate forms of hypertriglyceridemia are associated with both

increased hepatic production of triglyceride-rich lipoproteins (68-70) and impaired

metabolism and removal from plasma of apo B100-containing lipoproteins (71). In some

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hypertriglyceridemic patients, the production of hepatic triglyceride-rich lipoproteins was

found to vary in parallel with rates of bile acid synthesis (68-70,72). Reduced absorption of

bile acids displayed by type IV hypertriglyceridemic patients may be responsible for

increased bile acid synthesis (73). The linkage between the bile acid synthetic pathway and

hypertriglyceridemia becomes more apparent when type IV patients are treated with

cholestyramine, which both induces CYP7A1 expression and the hepatic production of

VLDL triglycerides (74). Conversely, feeding hypertriglyceridemia patients

chenodeoxycholic acid, which represses CYP7A1, reduces plasma triglyceride levels (75).

The recent finding that genetic loss the ileal bile acid receptor resulted in a familial form of

type IV hyperlipidemia (72) provides further evidence suggesting that stimulation of bile

acid synthetic pathway is involved. Our findings demonstrating that CYP7A1 transgenic

mice display a 2-fold increased production of apo B100/triglyceride lipoproteins but no

accumulation in plasma of triglyceride suggests that stimulation of the bile acid synthetic

pathway is not by itself sufficient to cause hypertriglyceridemia.

Our data suggest that SREBP-mediated gene expression links the anabolic VLDL

production pathway to the cholesterol/bile acid catabolic pathway through changes in

hepatic cholesterol levels and metabolism (19). The recent discovery that the nuclear

hormone receptors LXR and FXR are activated by oxysterols (76,77) and bile acids (44-46),

respectively provides an additional connection between CYP7A1 and VLDL production.

Many of the intermediates that are formed during the conversion of cholesterol to bile acids

are oxysterols that activate LXR (76,77). Moreover, the finding that LXR activates both

activates CYP7A1 (78) and SREBP1c expression (28,79), provides an additional

mechanism linking the bile acid biosynthetic pathway to the anabolic VLDL production

pathway. Our studies of CYP7A1 transgenic mice provide support linking

cholesterol/oxysterol metabolism to the hepatic expression of genes controlling lipoprotein

production and metabolism.

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Footnotes

*This work was supported by National Institute of Health grants HL57974 and HL51648.

1Abbreviations used are: apo, apolipoprotein; BSA, bovine serum albumin; cpm, counts per

minute; CYP7A1, cholesterol-7α-hydroxylase; FPLC, fast perfomance liquid

chromatograpghy; FXR, farnesoid X receptor; GAPDH, glyceraldehyde-3-phosphate-

dehydrogenase; HDL, high density lipoprotein; HMG-CoA reductase, 3-hydroxy, 3-

methyl-glutaryl-Coenzyme A reductase; HPLC, high pressure liquid chromotography; IDL,

intermediate density lipoprotein; kb, kilobase pair; LDL, Low density lipoprotein; LXR, liver

X receptor; MTP, microsomal triglyceride transfer protein; PCR, polymerase chain reaction;

PDI, protein disulfide isomerase; SCD-1, stearoyl CoA desaturase; SREBP, sterol response

element binding protein; TCA, trichloroacetic acid; VLDL, very low density lipoprotein.

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Figure legends

Figure 1. Transgenic mice express CYP7A1 mRNA (A), protein (B) and enzyme

activity (C) at constitutively high levels. Livers from transgenic and non-transgenic

mice fed a chow diet were obtained and used for the preparation of mRNA or microsomal

membrane fractions. (A) The expression of rat CYP7A1 transgene was used to examine

mRNA expression in mice that had previously been identified by PCR analysis as carrying

the transgene (+) or not (-). This RNase protection assay did not detect any rat CYP7A1

transgene expression in kidney, heart, lung and intestine (data not shown). (B) The indicated

amounts of protein from microsomes were electrophoresed through a 5-15% SDS-

polyacrylamide gel and transferred to a nitrocellulose blot. Western blot analysis with a

polyclonal antibody specific for rat CYP7A1 was used. This antibody also reacts with that

also detects mouse CYP7A1. (C) CYP7A1 enzyme activity from the microsomes of 3

transgenic and 3 non-transgenic mice.

Figure 2. Transgenic expression of CYP7A1 increases the bile acid pool size

The small intestine, liver and gall bladder were isolated from transgenic and non-transgenic

mice (3 per group N4 generation) and bile acid pool size and composition was determined

by HPLC (34). (T-Muri, tauromuricholic acid; T-Hyo, taurohyodeoxycholic acid; T-O,

taurine conjugates of minor species of bile acids; T-CA, taurocholic acid; T-CDCA,

taurochenodeoxycholic acid).

Figure 3. Northern blot analysis of various lipogenic genes expressed in CYP7A1

transgenic and non-transgenic mice. Poly (A) RNA was isolated from the livers of each

group of mice on a chow diet (4 per group left panel and 3 per group right panel) at mid-

light. The RNA was electrophoresed through a 0.8% agarose formaldehyde gel, transferred

to nylon and sequentially hybridized with the indicated radiolabeled cDNA probes for each

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panel. The expression level of the indicated genes is relative to GAPDH expression and

shown as the mean + S.D.

Figure 4. Transgenic expression of CYP7A1 increases the hepatic production and

secretion of Triglyceride. Triton WR-1339 was injected into the tail-vein of mice. At the

indicated time, blood was obtained from the retro-orbital sinus. (The content of triglycerides

was determined by an enzymatic assay. Each point represents the mean ± S.D. of 4 mice in

each group. *Denotes a significant difference (p<0.01) between non-transgenic (open bars)

and CYP7A1 transgenic (hatched bars) mice. The slope of the rate of increase in plasma

triglycerides was 2.2-fold greater in CYP7A1 mice than in non-transgenic littermates.

Figure 5. Transgenic expression of CYP7A1 increases the hepatic production and

secretion of [35

S] methionine-labeled de novo synthesized apo B100 and apo B48.

[35

S] methionine was injected intraperitoneally and 2 hours later Triton WR-1339 was

injected via the tail-vein. At the indicated time following Triton WR-1339 administration,

blood was collected by retro-orbital puncture into heparanized Natelson tubes. Aliquots of

plasma were subjected to SDS-PAGE. The portion of the gel containing apo B100 or apo

B48 was excised and solubilized with hyamine hydroxide then the amount of [35

S]

methionine quantitated by β-scintillation counting. At each time point, one µl of plasma was

placed on a filter paper, which was immersed in 15% trichloroacetic acid. The filter paper

was then washed with acetone and ethyl ether. The TCA-precipitated protein was then

subjected to β-scintillation counting. Each point represents the relative amount of [35

S]

methionine in apo B100 or apo B48 relative to the total [35

S] methionine-labeled de novo

synthesized plasma proteins (TCA precipitable protein). The slope of the linear rate of

accumulation in plasma of [35

S] methionine-labeled apo B100 or apo B48 relative to total

[35

S] methionine-labeled plasma proteins (determined by least squares analysis) is indicated

in each figure. The mean ± S.D. for the slopes of each group of mice are reported. (There

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were 4 individual mice in each group.) *Denotes a significant difference (between CYP7A1

transgenic mice and non-transgenic littermates) for the rate of accumulation of [35

S]

methionine-labeled apo B100 (p<0.025) and apo B48 (p<0.05) relative to total [35

S]

methionine-labeled plasma proteins.

Figure 6. Plasma triglyceride and apo B containing lipoprotein cholesterol in

transgenic and non-transgenic mice on a chow diet. Fasting plasma triglycerides (A)

and apo B containing lipoprotein cholesterol (VLDL, IDL, and LDL cholesterol) (B) were

measured by enzymatic assay. The mean + S.D. were 9 mice in each group are shown. (C)

FPLC analysis of plasma pooled from CYP7A1 4 transgenic and 4 non-transgenic mice.

(D) Plasma from CYP7A1 transgenic and non-transgenic mice were assayed for apo B100,

apo B48 and albumin by western blot analysis.

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Acknowledgements

Emma Du, Shui-Long Wang, Allis Ip, Monica Gaya are acknowledged for their

technical help and discussions.

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CYP7A1

β-actin

STD - - - - + + + + CYP7A1 transgene

CYP7A1 transgene + + + - - -

A B

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T-MCA T-HCA T-O T-CA T-CDCA

A BBiliary Bile Acid% Composition

0

50

100

150

200

250

300

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CYP7A1 Tg - - - + + +

MTP

Apo B

GAPDH

CYP7A1 Transgene

Mouse CYP7A1

1.0 ± 0.03

2.9 ± 0.5

Stearoyl CoA desaturase1

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Farnesyl diphosphate synthase

SREBP1

SREBP2

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Fatty acid synthase

GAPDH

LDL-receptor

HMG-CoA reductase

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4.3 ± 0.9

5.8 ± 0.9

5.9 ± 0.7

3.9 ± 0.3

4.9 ± 0.4

5.2 ± -0.6

CYP7B

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1.6 ± 0.5

3.2 ± 0.7

CYP7A1 Tg - - - + + +

SR-B11.1 ± 0.7

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100

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02 cp

m/µ

l)

Hours after Triton WR-1339

0

0.01

0.02

0.03

0.04

0.05

0.06

1 2 3 4

35S-

labe

led

apo

B10

0(1

02 cp

m/µ

l)

Hours after Triton WR-1339

1 2 3 40

0.030.050.070.090.11

0.150.170.190.21

0.13

Hours after Triton WR-1339

35S-

labe

led

apo

B48

(102

cpm

/µl)

1 2 3 40

0.050.070.090.110.130.150.170.190.210.23

Hours after Triton WR-1339

35S-

labe

led

apo

B48

(102

cpm

/µl)

Slope(102 cpm/µl/h)

Slope(102 cpm/µl/h)

Slope(102 cpm/µl/h)

Slope(102 cpm/µl/h)

0.0075 0.0063 0.0078 0.0077

.0073 ± .0007

0.0128 0.0141 0.0112 0.0121

.012 ± .001*

0.0505 0.0414 0.0390 0.0334

.0041 ± .007

0.052 0.056 0.054 0.044

.051 ± .005*

Non-Tg

Non-Tg

CYP7A1-Tg

CYP7A1-Tg

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A BPl

asm

a T

rigl

ycer

ide

(mg/

dl) 80

40

20

60

0CYP7A1 TgNon-Tg

D

CYP7A1 Tg

Non

-HD

L c

hole

ster

ol(m

g/dl

)

0

20

40

Non-Tg

*

C

Fraction Number

Apo B100

Apo B48

- - - - + + + + CYP7A1 Tg

Albumin

0

2

4

6

8

10

12

14

Cho

lest

erol

(mg/

dl)

CYP7A1 Tg

Non- Tg

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Castellani, Linda K. Curtiss, John M. Taylor and Roger A. DavisJon H. Miyake, Xuan Dao T. Doung, William Strauss, Gina L. Moore, Lawrence W.

-hydroxylaseαhyperlipidemia in transgenic mice expressing cholesterol-7Increased production of Apo B100-containing lipoproteins in the absence of

published online April 25, 2001J. Biol. Chem. 

  10.1074/jbc.M101853200Access the most updated version of this article at doi:

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Additions and Corrections

Vol. 276 (2001) 23304–23311

Increased production of apolipoprotein B-containing lipoproteins in the absence of hyperlipidemia in transgenicmice expressing cholesterol 7�-hydroxylase.

Jon H. Miyake, Xuan-Dao T. Doung, William Strauss, Gina L. Moore, Lawrence W. Castellani, Linda K. Curtiss, John M. Taylor,and Roger A. Davis

Page 23306: Fig. 1C has the wrong labels. The open bars designate the values for the non-transgenic mice, whereas the hatchedbars designate the values for the CYP7A1 transgenic mice.

Page 23310: The last sentence of the first paragraph should read: “Recent studies showed that treating rat hepatoma cells(McRH7777) with a 3-hydroxy-3-methylglutaryl-CaA reductase inhibitor selectively decreased SREBP1c mRNA induced by anoxysterol-mediated activation of liver X receptor (28).”

Vol. 276 (2001) 30490–30498

Directing the immune response to carbohydrate antigens.

Gina Cunto-Amesty, Tarun K. Dam, Ping Luo, Behjatolah Monzavi-Karbassi, C. Fred Brewer, Thomas C. Van Cott, and ThomasKieber-Emmons

Page 30493: The legend to Table I is incomplete. The correct table and legend are shown below.

Additional Refs. 66 and 67 are shown below Table I.

66. Oldenburg, K. R., Loganathan, D., Goldstein, I. J., Schultz, P. G., and Gallop, M. A. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 5393–539767. Scott, J. K., Loganathan, D., Easley, R. B., Gong, X., and Goldstein, I. J. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 5398–5402

TABLE IPeptides used in this study and their secondary structure properties

The secondary structure profiles were calculated from neural network calculations. The aromatic YPY motif-associated peptide mimetics 908 and912 were first identified from screening a random peptide library (Phage display) with ConA (66, 67). Arg-containing peptides representative ofthe WRYDI motif of peptide 107 were also identified, but these peptides were less effective inhibitors of MeaMan-ConA binding (66, 67) as ispeptide 107.

Number Sequence None All-� �/� All-�

105 GGIYYPYDIYYPYDIYYPYD ---EEE-------------- -------------------- -------------------- --EEEEEEEEEEEEEEE---107 GGIYYRYDIYYRYDIYYRYD ---EEEEEEEEEEEEEE--- -----H--H--H-------- ---E-EE-EE--H-EE---- --EEEEEEEEEEEEEEEE--911 YRYRYGRYRSGSYRYRYGRYRSGS ------------EEEE------- --------------H--------- -------------H---------- ---EE-EEE---EEEEE-EEE---908 DVFYPYPYASGS ------------ ------------ ------------ ---E--E-----909 DVFYRYRYASGS ---EEEE----- ---HHHHH---- ---EE-H----- --EEEEEE----910 DVFWRYRYASGS ---EEEE----- -----HHH---- ----EHHH---- ---EEEEE----712 MYWYPYASGS ---------- ---------- ---------- --EEEE----912 RYRYGRYRSGS ---------- ---------- ---------- ---EE--EEE---711 GGPGQPGQPGQPGQ ------------- -------------- -------------- --------------

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276, No. 44, Issue of November 2, p. 41526, 2001© 2001 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

We suggest that subscribers photocopy these corrections and insert the photocopies at the appropriateplaces where the article to be corrected originally appeared. Authors are urged to introduce thesecorrections into any reprints they distribute. Secondary (abstract) services are urged to carry notice ofthese corrections as prominently as they carried the original abstracts.

41526