J Biol Chem, Vol. 273, Issue 51, 34316-34327, December 18, 1998
cDNA Cloning of Mouse and Human Cholesterol 25-Hydroxylases,
Polytopic Membrane Proteins That Synthesize a Potent Oxysterol
Regulator of Lipid Metabolism*
Erik G.
Lund
,
Thomas A.
Kerr,
Juro
Sakai,
Wei-Ping
Li§, and
David W.
Russell¶
From the Departments of Molecular Genetics and
§ Cell Biology, University of Texas Southwestern Medical
Center, Dallas, Texas 75235-9046
 |
ABSTRACT |
Oxysterols regulate the expression of genes
involved in cholesterol and lipid metabolism and serve as intermediates
in cholesterol catabolism. Among the most potent of regulatory
oxysterols is 25-hydroxycholesterol, whose biosynthetic enzyme has not
yet been isolated. Here, we report the cloning of cholesterol
25-hydroxylase cDNAs from the mouse and human. The encoded enzymes
are polytopic membrane proteins of 298 and 272 amino acids,
respectively, which contain clusters of histidine residues that are
essential for catalytic activity. Unlike most other sterol
hydroxylases, cholesterol 25-hydroxylase is not a cytochrome P450, but
rather it is a member of a small family of enzymes that utilize diiron
cofactors to catalyze the hydroxylation of hydrophobic substrates. The
cholesterol 25-hydroxylase gene lacks introns, and in the human it is
located on chromosome 10q23. The murine gene is expressed at low levels in multiple tissues. Expression of cholesterol 25-hydroxylase in
transfected cells reduces the biosynthesis of cholesterol from acetate
and suppresses the cleavage of sterol regulatory element binding
protein-1 and -2. The data suggest that cholesterol 25-hydroxylase has
the capacity to play an important role in regulating lipid metabolism
by synthesizing a co-repressor that blocks sterol regulatory element
binding protein processing and ultimately leads to inhibition of gene transcription.
| |
INTRODUCTION |
Oxysterols are formed by the hydroxylation of the side chain of
cholesterol.1 This
modification renders the sterol more hydrophilic and confers two
important biological properties. First, the increased hydrophilicity enhances the ability of the oxysterol to cross membranes and
thereby facilitates its movement between intracellular compartments,
cells, and tissues. Second, oxysterols delivered in ethanol to cultured cells are potent regulators of the expression of genes involved in
sterol and fatty acid metabolism (1, 2).
The enhanced solubility of oxysterols is exploited by the body to
maintain cholesterol homeostasis. In several tissues and cell
types, including the brain, kidney, endothelium, and macrophages, cholesterol is converted into oxysterols that subsequently traverse the
plasma membrane and are transported to the liver (3-5). In the liver,
they are converted into bile acids by a newly described biosynthetic pathway (6). These bile acids are essential for normal
lipid and fat-soluble vitamin metabolism (7).
Oxysterols are both positive and negative regulators of gene
expression. As positive effectors, they bind to and activate the
nuclear receptor LXR (8), which in turn increases transcription of the
cholesterol 7
-hydroxylase gene (9). This activation stimulates the
conversion of cholesterol into bile acids (10). Mutation of the
LXR gene in mice causes a loss of 7-hydroxylase gene
induction and a build up of cholesterol in the liver (11). As negative
regulators, oxysterols suppress the cleavage of two transcription
factors known as sterol regulatory element binding proteins-1 and -2 (SREBP-1 and -2) (12). These proteins are synthesized as inactive
precursors in the membrane compartment of the cell. When intracellular
cholesterol levels decline, SREBPs are cleaved to release
amino-terminal fragments that migrate to the nucleus and activate the
transcription of a network of genes involved in cholesterol synthesis
and supply (12). This activation in turn restores intracellular
cholesterol levels.
Several oxysterols occur naturally, including
25-hydroxycholesterol (cholest-5-ene-3
,25-diol),
24-hydroxycholesterol (cholest-5-ene-3,24-diol), and
27-hydroxycholesterol (cholest-5-ene-3,27-diol) (13). Of these three
oxysterols, 25-hydroxycholesterol is the most potent regulator of
gene transcription when assayed in vitro (1, 2, 9, 11).
Despite this potency, it has proven difficult to document the
biosynthesis of this oxysterol and thus its role as an in
vivo regulator of lipid metabolism has remained questionable.
The objectives of the current study were to obtain in vivo
evidence for 25-hydroxylation of cholesterol and to identify the gene
responsible for this putative activity. We here report the isolation of
cDNAs encoding murine and human cholesterol 25-hydroxylases, and we
demonstrate that expression of these cDNAs in cultured cells
down-regulates cholesterol synthesis and SREBP processing.
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EXPERIMENTAL PROCEDURES |
Expression Cloning--
Total RNA was prepared from 400 mg of an
SREBP-1a transgenic mouse liver (14) using RNA-Stat 60 (Tel-Test, Inc.
Friendswood, TX). Poly(A)+ RNA was prepared from total RNA
by two cycles of chromatography on oligo(dT) (mRNA Purification
Kit, Amersham Pharmacia Biotech). A size-fractionated, directional
cDNA library with SalI and NotI cohesive ends
at the 5' and 3' termini, respectively, was constructed from 4 µg of
poly(A)+ RNA using a Superscript Plasmid Kit (Life
Technologies, Inc.). Size-fractionated cDNA (>1.0 kb, 10 ng) was
ligated with 50 ng of pCMV6 expression vector (a derivative of pCMV4
(15) containing a NotI site in the polylinker) using a
protocol and reagents supplied with the Superscript kit. Prior to
ligation, the pCMV6 vector (1.2 µg) was digested for 2 h with 10 units of NotI and SalI, respectively, in 30 µl
of 1× SalI restriction buffer (New England Biolabs,
Beverly, MA). The digested plasmid was purified by phenol/chloroform (1:1, v/v) extraction, electrophoresed on a 0.8% agarose gel, and
recovered from the gel using a QIAquick Gel Extraction Kit (Qiagen
GmbH, Germany).
Plasmid DNA was purified from the ligation reaction by precipitation
with ammonium acetate/ethanol and resuspended in 4 µl of water, of
which 1 µl was used to transform 40 µl of Electromax Escherichia coli DH10B cells (Life Technologies, Inc.). The
transformed bacteria were diluted into 1000 ml of LB medium containing
ampicillin. Aliquots of cells were plated on LB ampicillin plates for
calculation of the total number of recombinants. The remainder was
divided into 400 pools of 2.5 ml each that were grown to saturation
overnight at 37 °C. The total number of independent recombinants in
the cDNA library was 1.5 × 106, and each pool
contained an average of 3800 recombinants. DNA was prepared from
individual pools using a Wizard Miniprep Kit (Promega Inc, Madison,
WI). The yield of plasmid DNA from each pool was approximately 25 µg.
Human embryonic kidney 293 cells (ATCC CRL 1573) were plated on day 0 at a density of 7 × 105 cells/60-mm dish in Medium A
(Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf
serum, 100 units/ml penicillin, and 100 µg/ml streptomycin sulfate).
On day 1, individual dishes were transfected with a mixture of plasmid
DNAs that included pool DNA (5 µg), pCMV-StAR (2 µg), phct1 (2 µg), and pVA-1 (1 µg). The expression plasmid pCMV-StAR contains a
full-length cDNA encoding the murine steroidogenic acute regulatory
protein (StAR, Ref. 16) and was a kind gift of Dr. Douglas Stocco,
Texas Tech University Medical School, Lubbock, TX. The expression
plasmid phct1 contains a full-length cDNA encoding the murine
oxysterol 7-hydroxylase enzyme (17, 18). The original hct1 cDNA
was a kind gift of Dr. Richard Lathe, University of Edinburgh,
Edinburgh, Scotland. The plasmid pVA1 contains the adenovirus type 5 VAI gene (19). A positive control, in which cells were
transfected with 1 ng of a murine sterol 27-hydroxylase expression
plasmid diluted into 5 µg of pCMV6 vector alone, was included in
every experiment. Aliquots (40 µl) of the transfection lipid pfx-8
(Invitrogen, Carlsbad, CA) dissolved in DMEM containing 15 mM HEPES were added to each plasmid mixture. Cells were
incubated with the resulting lipid/DNA mixture in 5 ml of DMEM for
4 h at 37 °C, in an atmosphere of 8.8% CO2. Assay
of cholesterol 25-hydroxylase activity was thereafter carried out as
described below.
To subdivide the positive pool containing a cholesterol 25-hydroxylase
cDNA, an aliquot (40 µl) of Electromax DH10B cells was
transformed with 0.5 ng of DNA from the positive primary pool. A
portion (0.5%) of the transformation mixture was diluted into 50 ml of
LB medium containing ampicillin and divided into 20 pools, each
containing ~490 recombinants for secondary screening. DNA was
prepared from individual pools, and 293 cells were transfected as
above, except that 6-well plates were used in place of 60-mm dishes,
and amounts of reagents were scaled down accordingly. Tertiary
screening was similarly performed except subpools of 50 recombinants
were transfected. Quaternary screening was carried out with pools of 10 cDNA isolates derived from a matrix array of individual cDNAs
to identify a single cholesterol 25-hydroxylase cDNA.
Measurement of Cholesterol 25-Hydroxylase Activity in Whole
Cells--
The transfection medium containing the cationic lipid was
aspirated and replaced with 3 ml of Medium B (DMEM containing 10% newborn calf lipoprotein-poor serum, 100 units/ml penicillin, and 100 µg/ml streptomycin sulfate) supplemented with 5 µl of [4-14C]cholesterol (56.6 mCi/mmol; 0.040 µCi/µl; NEN
Life Science Products). Cells were incubated for a further 60 h at
37 °C in an atmosphere of 8.8% CO2.
Media from the transfected cells were collected and extracted with 8 ml
of chloroform/methanol (2:1, v/v). The organic phase from each sample
was taken to dryness under a stream of nitrogen, and residues were
dissolved in 40-µl aliquots of chloroform/methanol (2:1, v/v) and
applied to 20 × 20 cm prescored LK5DF silica gel TLC plates
(Whatman, Hillsboro, OR) with preadsorbent layers. The plates were
developed in ethyl acetate/toluene (4:6, v/v) and exposed to a Fuji
BAS-MP phosphorimager plate overnight. Phosphorimage analysis was then
performed on a Fuji BAS1000 apparatus.
Isolation of Human Cholesterol 25-Hydroxylase cDNA--
A
372-base pair expressed sequence tag (EST, GenBankTM
accession number W01328) with high sequence identity to a portion of
the murine 25-hydroxylase cDNA was identified by BLAST search. A
bacterial stab transformed with a plasmid containing the EST sequence
cloned into the pT3T7 vector was obtained from Research Genetics, Inc.,
Huntsville, AL. Plasmid DNA was prepared, and a 245-base pair fragment
was amplified by the polymerase chain reaction using oligonucleotide
primers of the following sequence: 5'-CTGGGACCACCTGAGGAGCT-3'
(forward primer) and 5'-GCCCAATGCAGCAGCGTCAC-3' (reverse primer).
The thermocycler program consisted of 35 cycles of 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s. The amplified cDNA fragment was cloned into pGEM-T Easy (Promega Corp., Madison, WI). The insert was excised from the plasmid with EcoRI and
used for the preparation of a radiolabeled probe by random octamer priming (Megaprime Labeling Kit, Amersham Pharmacia Biotech). The probe
was used to screen 200,000 plaques of a human lung cDNA library in
bacteriophage 
gt10 (catalog number HL3004a,
CLONTECH, Palo Alto, CA) using standard
hybridization procedures (20). One positive clone was isolated whose
cDNA insert was subcloned into the EcoRI sites of
pBluescript SK+ (Stratagene Corp., La Jolla, CA) and pCMV6,
yielding plasmids pBS-h25 and pCMV-h25, respectively.
Gene Mapping--
Cholesterol 25-hydroxylase gene sequences were
isolated from a murine genomic library prepared from 129SvEv DNA (a
kind gift of Dr. Alan Bradley, Baylor College of Medicine, Houston, TX) and a human genomic library (catalog number 946204, Stratagene), both
in bacteriophage FIX II, by screening with full-length cDNA
probes corresponding to the murine and human cholesterol 25-hydroxylase
cDNAs, respectively, using standard protocols (20). Approximately
600,000 murine and 400,000 human recombinants were screened, and
one positive clone from each library was identified and purified to
homogeneity. The corresponding genomic DNA inserts were excised from
the bacteriophage vectors and ligated into the NotI site of
pBluescript SK+, yielding plasmids pBS-mg25 and pBS-hg25, respectively.
The chromosomal location of the human cholesterol 25-hydroxylase gene
was determined by fluorescent in situ hybridization (FISH)
and by polymerase chain reaction amplification of somatic cell and
radiation hybrid panel DNAs. FISH mapping was performed by See DNA
Biotech, Inc. (Downsview, Ontario, Canada). The bacteriophage clone
harboring the human 25-hydroxylase gene described above was labeled
with biotinylated dATP for use as a FISH probe. Of 100 mitotic figures
analyzed, 91 showed hybridization signals on paired sister chromatids
corresponding to chromosome 10. Comparison of the signal positions with
bands generated by staining with 4,6-diamidino-2-phenylindole indicated
that hybridization occurred at band q23. Radiation and somatic cell
hybrid mapping were performed using DNAs in the Somatic Cell Hybrid
Mapping Panel 2 (Coriell Institute of Medical Research, Camden, NJ) and
the Stanford G-3 radiation hybrid panel (Research Genetics, Huntsville,
AL). The primer pair used for amplification was
5'-CTGGGACCACCTGAGGAGCT-3' (forward primer) and
5'-GCCCAATGCAGCAGCGTCAC-3' (reverse primer), which,
respectively, correspond to nucleotides 79-98 and 333-314 of the
human gene sequence (Fig. 6A). The thermocycler program consisted of 35 cycles of 94 °C for 15 s and 68 °C for
30 s on a Perkin-Elmer GeneAmp 9600 machine. Only somatic cell
hybrid DNAs containing human chromosome 10 produced a positive
amplification signal. Analysis of the radiation hybrid data through the
Stanford Genome Center
server2 indicated linkage of
the cholesterol 25-hydroxylase gene to the SHGC-15188 marker (LOD
score = 4.4, cR_1000 = 45.76) on chromosome 10 in the
vicinity of band q23.
DNA Sequencing and RNA Blotting--
DNA sequencing was
performed on an ABI Prism 377 sequencer using thermocycler sequencing
protocols and fluorescent dye terminators. Contiguous DNA sequences
were assembled using MacVector software (IBI-Kodak Corp., New Haven,
CT), and sequence alignments were generated using a Lasergene software
package (DNASTAR, Inc., Madison, WI).
For RNA blotting, a murine multiple tissue RNA blot
(CLONTECH, catalog number 7762-1) was hybridized
overnight in 50% formamide hybridization buffer at 42 °C with a
full-length murine 25-hydroxylase cDNA probe using standard
procedures (20). The probe was radioactively labeled by random nonamer
priming with [32P]CTP. The blot was washed stringently at
65 °C, in 0.1× SSC containing 0.1% (w/v) SDS before exposure for 5 days to Kodak X-OMAT AR film at 
80 °C using an intensifying screen.
Antibodies--
An antipeptide antibody against the sequence
RRYKIHPDFSPSVKQ, representing amino acids 69-83 of the murine
cholesterol 25-hydroxylase (Fig. 3A), was raised in rabbits.
This sequence was synthesized as a multiple antigen peptide by
Bio-Synthesis, Inc. (Lewisville, TX). For the initial immunization, 100 µg of peptide was administered intramuscularly as a dispersion in
Freund's complete adjuvant to two New Zealand White male rabbits, 3 months of age. Boosts of 100 µg of antigen in Freund's incomplete
adjuvant were given on average every 5 weeks, and bleeds were drawn 7 days after each boost. One of the two resulting antisera, U104, was
used here after affinity purification on peptide antigen columns
(21).
Epitope Tagging--
To construct an epitope-tagged version of
the murine cholesterol 25-hydroxylase enzyme, a cDNA fragment
spanning the coding region and having BspDI and
XbaI restriction sites at the 5' and 3' ends, respectively,
was amplified by the polymerase chain reaction using the
oligonucleotide primers 5'-AAAATCGATGGCTGCTACAACGGTTCGGA-3' (forward primer) and 5'-AAATCTAGATCAAGTCTGTTTCTTCTTCTGGATCA-3' (reverse primer). The template was the plasmid pCMV-m25, and the thermocycler program consisted of 35 cycles of 94 °C at 30 s, 57 °C at 15 s, and 72 °C at 60 s. The amplified
cDNA fragment was purified on a Centricon-100 column (Amicon Corp.,
Beverly, MA), digested with BspDI and XbaI,
repurified by chromatography on a Centricon-100 column, and ligated
into a modified pcDNA3 vector containing a sequence for a c-Myc
epitope in front of the BspDI site. The desired recombinant
was termed pcDNA3-NH2-myc-m25. This plasmid encodes a
fusion protein in which the sequence MEQKLISEEDLNEQKLISEEDLNID containing two tandem copies of a c-Myc epitope is linked to the amino
terminus of the murine cholesterol 25-hydroxylase protein lacking the
initial methionine residue. A similar strategy was used to place tandem
c-Myc epitopes at the amino terminus of the human cholesterol
25-hydroxylase cDNA, producing the plasmid
pcDNA3-NH2-myc-h25.
A double c-Myc epitope (EQKLISEEDLNEQKLISEEDLN) was placed at the
carboxyl terminus of the murine cholesterol 25-hydroxylase cDNA as
follows. An oligonucleotide encoding these epitopes with an
RsrII cohesive 5'-end and a blunt 3'-end was formed by
annealing the phosphorylated oligonucleotide primers
5'-GTCCGCGGAGCAAAAGCTCATTTCTGAAGAGGACTTGAATGAGCAAAAGCTCATTTCTGAAGAGGACTTGAATTAG-3' and
5'-CTAATTCAAGTCCTCTTCAGAAATGAGCTTTTGCTCATTCAAGTCCTCTTCAGAAATGAGCTTTTGCTCCGCGG-3' as described (22). The annealed duplex was ligated into pCMV-m25 that had been digested with Eco47III and RsrII
and purified by agarose gel electrophoresis. The resulting plasmid,
pCMV-m25-COOH-myc, encodes a protein comprising amino acids 1-267 of
the murine cholesterol 25-hydroxylase fused to the two c-Myc epitope sequences.
Mutagenesis--
Site-directed mutagenesis (23) was carried out
using a polymerase chain reaction-based kit (Quik-Change, Stratagene)
on the plasmid pCMV-m25. The mutagenic oligonucleotide primers
(5'-GGCTCACCACGACATGCAACAATCTCAGTTTAACTGC-3' and
5'-GCAGTTAAACTGAGATTGTTGCATGTCGTGGTGAGCC-3') were designed to convert
histidine codons at positions 242 and 243 of the murine protein to
glutamine codons. Mutagenesis was carried out according to instructions
provided by the manufacturer. Plasmid DNA products were subjected to
DNA sequence analysis to confirm the presence of the substitution
mutations and the absence of spurious mutations. The plasmid containing
the desired mutations was named pCMV-m25-HH242QQ.
Measurement of Cholesterol 25-Hydroxylase Activity in Cell
Lysates--
On day 0, a derivative of Chinese hamster ovarian cells
expressing the polyoma virus middle T antigen (31) (CHOP cells) was
plated at a density of 750,000 cells/100-mm dish in Medium C (1:1 (v/v)
DMEM/Ham's F12 medium containing 5% fetal calf serum, 100 units/ml
penicillin, and 100 µg/ml streptomycin sulfate). On day 1, the cells
were transfected with 1.5 µg of pVA-1 and 13.5 µg of pCMV6,
pCMV-m25-HH242QQ, or pCMV-m25 per dish for vector, mutant, and wild
type 25-hydroxylase transfections, respectively. 60 µl of pfx-8 lipid
was used as a transfection reagent as described above. On day 2, cells
were incubated for 1 h with 2% (w/v)
2-hydroxypropyl--cyclodextrin dissolved in a 1:1 solution of
DMEM/Ham's F12 medium. The cells were washed once with ice-cold PBS
and then harvested in the same buffer using a rubber policeman. After
centrifugation at 1000 × g for 5 min, the buffer was
aspirated, and the cell pellet was resuspended in 1 ml of 50 mM potassium phosphate buffer, pH 7.4, containing protease
inhibitors (Boehringer Mannheim Complete Mini, EDTA-free, at the
concentration recommended by the supplier). A cell lysate was prepared
using a Polytron set at 10,000 rpm, with three bursts of 3 s each
with 30-s intervals between bursts. Incubations were performed at
37 °C with 140 µg of cell lysate protein in 50 mM
potassium phosphate buffer, pH 7.4, containing 5 mM NADPH.
[4-14C]Cholesterol was added in 4 µl of 45% (w/v)
2-hydroxypropylcyclodextrin in water to a final concentration of 5 µM. The total volume of the incubation was adjusted to
200 µl. After 2 h, reactions were extracted with
chloroform/methanol (2:1, v/v) and analyzed by thin layer chromatography.
SREBP Cleavage Assay--
Stock cultures of CHO-7 cells, a
subline of CHO-K1 cells selected for growth in lipoprotein-deficient
serum (25), were maintained in Medium D (1:1 (v/v) DMEM/Ham's F12
medium containing 5% newborn calf lipoprotein-poor serum, 100 units/ml
penicillin, and 100 µg/ml streptomycin sulfate). On day 0, cells were
plated at a density of 7 × 105 cells/60-mm dish. On
day 1, transfections were carried out using 4 µg/dish of the
indicated plasmid DNA and LipofectAMINE reagent (Life Technologies,
Inc.) according to the manufacturer's instructions and with
modifications as described (26). After transfection, fresh media
supplemented with 50 µM mevalonate, 50 µM
compactin, and 0.2% ethanol containing either no sterol or a mixture
of sterols (final concentrations of 1 µg/ml 25-hydroxycholesterol and
10 µg/ml cholesterol) were added. The cells were returned for 20 h to a 37 °C incubator, harvested, and then fractionated into a
nuclear extract and a 105 × g membrane pellet as described
(27). Immunoblot analyses of SREBP-1 and -2 proteins were performed
using a SuperSignal Substrate kit (Pierce) and the murine monoclonal
antibodies IgG-2A4 and IgG-7D4 (28, 29).
Gas Chromatography-Mass Spectrometry--
Six-well plates of
CHOP cells were transfected with pCMV-m25 or with vector alone as
described above and incubated for 48 h in Medium D supplemented
with 10 µg/ml cholesterol. Thereafter, media were extracted with
chloroform/methanol (2:1, v/v; 5 ml/well), and the organic phase was
separated and taken to dryness under a stream of nitrogen. Extracts
from 6 wells were combined for subsequent procedures. The samples were
purified on Isolute Silica columns (International Sorbent Technology,
Mid Glamorgan, UK), and hydroxyl groups were converted to
trimethylsilyl ethers as described previously (13).
Gas chromatography-mass spectrometry was performed on a Varian 3400 gas
chromatograph equipped with an HP-5MS capillary column (30 m × 0.25 mm, 0.25-µm phase thickness) connected to a Finnigan SSQ700 mass
spectrometer. The gas chromatography temperature program was 180 °C
for 1 min, followed by a temperature gradient of 10 °C/min to
300 °C, and a final elution at 300 °C for 15 min. Helium was used
as the carrier gas at an injector valve pressure of 6 pounds/square
inch. Injector and transfer line temperatures were set to 280 °C,
and the injector was operated in the splitless mode. The machine was
operated in the electron ionization mode with electron energy set to 70 eV, and the quadrupole was scanned between m/z 100 and 500 at a rate of 1 scan/1.5 s.
Analysis of N-Linked Carbohydrates--
To examine the
sensitivity of N-linked carbohydrates on cholesterol
25-hydroxylase to endoglycosidase digestion, COS M6 cells were
initially plated at a density of 5 × 105 cells/60-mm
dish in Medium A on Day 0 of the experiment. On Day 1, one dish each
was transfected with 4.5 µg of pCMV-m25,
pcDNA3-NH2-myc-m25, pCMV-m25-COOH-myc, pCMV-h25, or
pcDNA3-NH2-myc-h25 together with 0.5 µg of pVA1 and 20 µl of pfx-8 lipid as described above. After transfection, cells were
cultured in Medium B. On day 2, cells were harvested using a rubber
policeman, pelleted at 1000 × g, washed with 1 ml of
phosphate-buffered saline, pH 7.4, and resuspended in 0.2 ml of a
buffer containing 10 mM Tris-Cl, pH 8.0, and 1 mM EDTA. Cells were lysed by 20 passages through a 22-gauge
needle, and aliquots of the lysates (10 µl, ~40 µg of protein)
were treated with endoglycosidase H or peptide N-glycosidase
F overnight in a volume of 30 µl according to the instructions given
by the supplier (New England Biolabs, Beverly, MA). One volume of 2×
Laemmli gel loading buffer was then added to each sample, followed by
incubation at 100 °C for 10 min and electrophoresis through a 12%
SDS-polyacrylamide gel for 16 h at constant current (10 mA).
Separated proteins were electroblotted to polyvinylidene difluoride
membranes (30), which were incubated with affinity purified (21)
antipeptide antibody directed against cholesterol 25-hydroxylase
(U-104, see above) at 0.8 µg/ml. A goat anti-rabbit horseradish
peroxidase-conjugated antibody (Amersham Pharmacia Biotech) was used as
secondary antibody, and visualization was via an ECL Plus kit (Amersham
Pharmacia Biotech).
Cytochemistry--
For indirect immunocytochemistry, COS M6
cells were plated at a density of 4 × 104 cells per
well on glass coverslips placed in 6-well dishes containing Medium B. On Day 1, cells were transfected with either a vector alone control
(pCMV6) or with pCMV-m25-COOH-myc-m25 or
pcDNA3-NH2-myc-m25. Three µg of plasmid DNA and 12 µl of pfx-8 lipid were used per well. After transfection, cells were
cultured in Medium B. Indirect immunocytochemistry was then performed
with the indicated antibody and lectin probes as follows. Cells were
fixed for 30 min with 3% (w/v) paraformaldehyde in Hanks' balanced
salt solution, pH 7.4. Following fixation, the coverslips were briefly
rinsed with PBS (0.1 M phosphate buffer, pH 7.4, 0.15 M NaCl), and free aldehyde groups were quenched by
incubation in PBS containing 50 mM NH4Cl for 30 min. Permeabilization was accomplished by incubation in 0.1% (v/v)
Triton X-100 in H2O for 7 min on ice, followed by rinsing in PBS containing 1% (w/v) bovine serum albumin (blocking buffer) for
30 min at room temperature. Coverslips were incubated with rabbit
anti-c-Myc IgG (Upstate Biotechnology Inc., 10 µg/ml in blocking
buffer) for 2 h at room temperature. Finally, coverslips were
incubated with fluorescein isothiocyanate goat anti-rabbit IgG
(Zymed Laboratories Inc.; 20 µg/ml in blocking
buffer) for 1 h at room temperature. For Golgi compartment
staining, rhodamine-labeled wheat germ agglutinin was added during the
second antibody incubation at a concentration of 1.25 µg/ml.
Coverslips were washed three times with PBS containing 0.1% bovine
serum albumin after each antibody or lectin incubation. Cells were
photographed using a Zeiss Photomicroscope.
Inhibitor Studies--
Transfection of CHOP cells was carried
out as described above with the following exceptions. Cells were plated
on Day 0 at a density of 150,000 cells/well in 6-well plates containing
Medium C. On Day 1, cells were transfected with 2.7 µg of the
indicated cholesterol 25-hydroxylase expression plasmid and 0.3 µg of
pVA-1, using 12 µl of pfx-8 as transfection lipid. After 4 h,
the lipid/DNA mixture was removed, and 1.5 ml of Medium D was added. In
certain experiments, this medium was aspirated and replaced on Day 2 with 1 ml per well of a 20 mg/ml solution of
2-hydroxypropyl--cyclodextrin (Sigma) in DMEM/Ham's F12 (1:1)
medium and returned to the incubator. The cyclodextrin-containing
medium was replaced after 1-1.5 h with 1.5 ml of Medium D, and
substrate and inhibitors were then added, each in a volume of 4.5 µl
of ethanol. The concentration of [4-14C]cholesterol
substrate (specific activity = 26.8 mCi/mmol) was 3 µM. Inhibitors were added to final concentrations of 3, 10, or 30 µM as indicated in Fig. 9. Cells and/or cells
plus medium were harvested at the indicated times, extracted, and the
conversion of substrate into [4-14C]25-hydroxycholesterol
product was determined by thin layer chromatography as described above.
The inhibitors used were cholesterol (5-cholesten-3-ol), cholestanol
(5-cholestan-3-ol), epicholesterol (5-cholesten-3-ol), coprostanol (5-cholestan-3-ol), desmosterol (5, 24-cholestadien-3-ol), -sitosterol
(5-cholesten-24-ethyl-3-ol), 25-hydroxycholesterol (cholest-5-ene-3, 25-diol), and 27-nor-25-oxocholesterol
(27-nor-25-oxo-5-cholesten-3-ol). All steroids were purchased from
Steraloids Inc. (Wilton, NH), except cholesterol, which was from Sigma.
Stable Cell Lines--
EcR-CHO cells (Invitrogen), a cell line
stably expressing the subunits of the Drosophila ecdysone
receptor, RXR and VgECR (31), were plated on day 0 at a density of
500,000 cells/100-mm dish in Medium C supplemented with 250 µg/ml
Zeocin. On day 1, cells were transfected with 5 µg of pIND-m25 using
15 µl of Fugene 6 (Boehringer Mannheim) according to the instructions
of the manufacturer. The plasmid pIND-m25 was constructed by insertion
of a full-length mouse 25-hydroxylase cDNA fragment from plasmid
pCMV-m25 into the pIND vector (Invitrogen). On day 2, cells were split
1:15 and plated in fresh Medium C supplemented with 250 µg/ml Zeocin and 700 µg/ml geneticin. Cells were refed this medium every 2nd day,
and on day 10, groups of five geneticin-resistant colonies were
replated in individual wells. Following expansion, cells were tested
for cholesterol 25-hydroxylase expression by addition of ponasterone
(Invitrogen) to a final concentration of 5 µM (16 h),
followed by immunoblot analysis of total cell protein. Antibody U104
(affinity purified) was used to detect expression of the enzyme as
described above. One positive group of cells was selected and subcloned
through one additional round to ensure clonality. One of the resulting
cell strains, designated TR3102a, which manifests high level expression
of cholesterol 25-hydroxylase upon ponasterone induction, and another,
designated TR3102g, with no detectable inducible enzyme expression,
were selected and maintained as lines. For routine induction
experiments, 10 µM ponasterone was used for the indicated periods.
Cholesterol Biosynthesis--
TR3102a and TR3102g cells were
plated on day 0 at a density of 250,000 cells/60-mm dish in Medium C
containing Zeocin and geneticin as above. On day 1, the medium was
changed to Medium F (Medium D containing 250 µg/ml Zeocin, 700 µg/ml geneticin, and 10 µM ponasterone). On day 2, 20 µl of an aqueous solution containing 15 µCi of
[1,2-14C]acetate (American Radiolabeled Chemicals),
adjusted with cold acetate to a final mass of 1 µmol, was added to
each dish. The additions were made in a staggered fashion so that all
cells were harvested at the same time, corresponding to incubation
times of 2, 4, and 6 h, respectively. The total time of induction
with ponasterone was 27 h, including the acetate labeling period.
Nonsaponifiable lipids were isolated and analyzed by thin layer
chromatography as described (32) except that 5 µCi of
[26,27-3H]25-hydroxycholesterol (NEN Life Science
Products) was used as a standard. Quantification of acetate
incorporation into cholesterol was via phosphorimage analysis.
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RESULTS |
The livers of transgenic mice overexpressing the transcription
factor SREBP-1a accumulate large quantities of cholesterol and
triglycerides, owing to the overproduction of lipid synthesizing enzymes (14). An analysis of stool lipids by mass spectrometry revealed
that these animals also excrete high levels of several oxysterols,
including 25-hydroxycholesterol. To isolate cDNAs that encode
putative oxysterol synthesizing enzymes from the livers of these
transgenic mice, an expression cloning strategy in cultured mammalian
cells was conceived and optimized. The basic premise of the screen was
to transfect cells with pools of hepatic cDNAs cloned into an
expression vector, add [14C]cholesterol to the medium,
and then measure the conversion of this substrate into oxysterols by
thin layer chromatography assay. Initially, we used a previously
isolated sterol 27-hydroxylase cDNA, whose encoded enzyme converts
cholesterol into the oxysterol 27-hydroxycholesterol (15), to optimize
assay parameters.
Chromatography studies with oxysterol standards revealed that the
separation between cholesterol and some oxysterols was poor on silica
gel plates. Furthermore, in control transfection studies with the
sterol 27-hydroxylase expression vector, the strong phosphorimage signal from the substrate often obscured a weaker product signal. To
overcome these problems, a cDNA encoding a murine oxysterol 7-hydroxylase (18) was cotransfected into the cells. This addition should result in the conversion of oxysterol products to their 7-hydroxylated forms. The oxysterol 7-hydroxylase also possesses a minor 2-hydroxylase activity against 7-hydroxylated sterols (18);
thus the formation of 2,7-hydroxylated oxysterols was expected. Both
of these classes of hydroxylated oxysterols were readily separated from
cholesterol by thin layer chromatography. The optimum transfection host
(293 cells), transfection method (lipofection with pfx-8 lipid), and
transient expression time (60 h) were determined. Further experiments
confirmed that the sterol 27-hydroxylase enzyme, which is located in
the mitochondria (15), was stimulated 2-3-fold when a cDNA
encoding the murine steroidogenic acute regulatory protein was
cotransfected into cells (16, 33). Finally, addition of the adenovirus
VA1 gene, which enhances the translation of mRNAs
transcribed from transfected plasmids (19), to the DNA mixture
stimulated expression levels another 1.5-fold. Under these optimized
conditions, sterol 27-hydroxylase enzyme activity could be detected
over background when the cDNA expression vector was diluted
3,000-5,000-fold.
A library consisting of 1.5 × 106 individual
cDNAs was next constructed in a pCMV6 vector using
poly(A)+ mRNA isolated from an SREBP-1a transgenic
mouse liver. Two hundred fifty-five aliquots of ~3,800 individual
plasmids from the library were screened using the optimized parameters
described above. The data of Fig. 1 show
the results of a transfection experiment that revealed several positive
cDNA pools. The pool analyzed in lane 1 did not contain
a cDNA encoding a cholesterol-metabolizing enzyme. However, the
pools analyzed in lanes 2-5 produced low to very low levels
of two sterols that migrated more slowly and thus were more hydrophilic
than the cholesterol substrate. Additional experiments revealed that
the pools analyzed in lanes 2-4 contained sterol
27-hydroxylase cDNAs, whereas that analyzed in lane 5 contained a cholesterol 25-hydroxylase cDNA (data not shown).
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Fig. 1.
Expression cloning of oxysterol
cDNAs. Mixtures of expression plasmids specifying the murine
steroid acute regulatory protein (2 µg), murine oxysterol
7-hydroxylase (2 µg), adenovirus VA-1 RNA (1.0 µg), and pools of
3,000-4,000 independent isolates from an SREBP-1a transgenic mouse
liver cDNA library (5 µg) were transfected into 60-mm dishes of
cultured human embryonic kidney 293 cells and assayed for their ability
to convert [14C]cholesterol (0.7 µM) to
oxysterols. Enzyme activities encoded by five representative pools
of cDNAs are shown in a phosphorimage derived from a thin layer
chromatogram. The positions to which the cholesterol substrate and
oxysterol products migrated to on the silica gel plate and their
chemical identities are shown on the right. The pools
assayed in lanes 2-4 contained cDNAs encoding sterol
27-hydroxylase. The pool assayed in lane 5 contained a
cholesterol 25-hydroxylase cDNA. The primary and secondary 25- and
27-hydroxylated products arising from the expression of these two
enzymes comigrate in the solvent system employed.
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The pool containing the 25-hydroxylase cDNA was progressively
subdivided and expressed to isolate a single cDNA. As the purity of
the cDNA increased, the level of product generated in the
transfected cells also increased to the point that cotransfection of
the oxysterol 7-hydroxylase cDNA was dispensable. Additional
experiments revealed that the cDNA-encoded enzyme was not
stimulated by inclusion of the steroidogenic acute activator cDNA,
suggesting that it was not a mitochondrial protein. Transfection of the
pure cDNA into CHOP cells produced abundant 25-hydroxylase enzyme
activity that increased with time of incubation (Fig.
2A). The activity was stimulated approximately 10-fold by treatment of transfected cells with
2-hydroxypropyl--cyclodextrin (Fig. 2A). This compound
presumably removes endogenous cholesterol from the membranes of the
transfected cells that otherwise competes with the exogenously added
radiolabeled cholesterol substrate (34).
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Fig. 2.
Expression of cholesterol 25-hydroxylase
enzyme activity in transfected cells and stimulation by cyclodextrin
treatment. A, cultured CHOP cells were transiently
transfected with the murine or human cDNA expression plasmid on Day
1 of the experiment. On day 2, the indicated dishes were treated for
1 h with 20 mg/ml 2-hydroxypropyl--cyclodextrin dissolved in
DMEM/Ham's F12 (1:1) medium. This solution was aspirated, and fresh
media containing 3.0 µM [14C]cholesterol
was added to all dishes, and the incubation was continued for an
additional 4-24 h. Sterols were extracted, and the conversion of
[14C]cholesterol into
[14C]25-hydroxycholesterol was determined by thin layer
chromatography assay. An autoradiogram derived from the silica gel
plate is shown with the positions of cholesterol,
25-hydroxycholesterol, and sterol esters marked on the left.
The percent conversion of substrate into product determined by
quantification of the phosphorimage analysis is indicated
below each lane. B, chemical analyses of
cholesterol 25-hydroxylase products. Sterols from the media of cells
transfected for 4.0 h with plasmid vector alone or vector
containing a murine cholesterol 25-hydroxylase cDNA were
extracted with organic solvent, derivatized, and subjected to gas
chromatography-mass spectrometry. A cDNA-dependent product
eluting at 23.56 min from the gas chromatograph (left
panel) had an ionization spectrum virtually identical to
that of authentic 25-hydroxycholesterol (right
panels).
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The chemical structure of the oxysterol produced by the isolated
cDNA was determined by gas chromatography-electron ionization mass
spectrometry (Fig. 2B). The media from cells transfected with the putative cholesterol 25-hydroxylase cDNA contained a prominent sterol eluting at 23.56 min from the gas chromatography column. This sterol was not present in the media of mock-transfected cells (Fig. 2B, left panels). The mass
spectrum of the cDNA-generated product was virtually identical to
that of an authentic 25-hydroxycholesterol standard (Fig.
2B, right panels).
A search of the DNA data bases revealed a human EST with identity
to the murine 25-hydroxylase cDNA. This EST was used to isolate a
near full-length cDNA encoding the human 25-hydroxylase as
described under "Experimental Procedures." Transfection into 293 cells of the human cDNA cloned in a pCMV6 vector produced abundant
25-hydroxylase enzyme activity (Fig. 2A). This activity was
stimulated approximately 5-fold by treatment of cells with 2-hydroxypropyl-cyclodextrin (Fig. 2A).
Fig. 3A shows an alignment of
the deduced amino acid sequences of the murine and human cholesterol
25-hydroxylases. The two proteins share 78% sequence identity (Fig.
3A), whereas the encoding cDNAs are 82% identical in
their translated regions. The predicated molecular weights of the
murine and human enzymes are 34,700 and 31,700, respectively. The most
notable difference between the two is a 26-amino acid extension at the
carboxyl terminus of the murine enzyme that is not present in the human
enzyme (Fig. 3A). Both proteins have three clusters of
conserved histidine residues (amino acids 143-147, 157-161, and
238-243 in the murine sequence, Fig. 3A). Similar clusters
of histidine residues are present in a Pseudomonas alkane
hydroxylase and xylene monooxygenase (35, 36), the eukaryotic
stearoyl-CoA desaturases (37), and the yeast and human C-4 sterol
methyl oxidases (38, 39). These enzymes are members of a family of
proteins that utilize diiron cofactors to catalyze diverse reactions on
hydrophobic substrates. Hydropathy analyses of the 25-hydroxylase
sequences revealed four conserved regions of extended hydrophobicity
that may constitute as many as eight transmembrane domains (Fig.
3B). The location of the first, second, and fourth of these
hydrophobic regions coincided with similar sequences in the
Pseudomonas alkane hydroxylase, which contains six
transmembrane domains (40).
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Fig. 3.
Structure of murine and human cholesterol
25-hydroxylases. A, line up of the cDNA-deduced
protein sequences of the murine and human enzymes. Individual amino
acids are shown in single letter code. Sequence identities
are indicated by the black boxes. Amino acids are
numbered on the right. The GenBankTM
accession numbers for the murine and human cDNA sequences are
AF059213 and AF059214, respectively. B, hydropathy analyses
comparing the primary sequences of the murine cholesterol
25-hydroxylase (upper panel) and the human
cholesterol 25-hydroxylase (lower panel).
Predicted hydrophobic regions of the protein sequences fall below the
midlines, whereas hydrophilic sequences rise above the midlines.
MacVector software was used to generate the data. The window size in
the scan was 12 amino acids.
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To determine if the clustered histidine residues in cholesterol
25-hydroxylase were important for enzyme activity, a pair of histidine
codons at positions 242 and 243 in the murine protein were changed to
glutamine codons by site-directed mutagenesis of the wild type
cDNA. The resulting mutant cDNA was transfected into CHOP cells
and assayed for expression of the protein and for cholesterol
25-hydroxylase enzyme activity (Fig.
4A). Mutation of the two
histidine residues had no effect on steady state expression levels as
judged by immunoblotting (Fig. 4A, left
panel) but eliminated enzyme activity in transfected cells
(Fig. 4A, right panel). Similar results were obtained in a second experiment in which cholesterol 25-hydroxylase enzyme activity was measured in cell lysates rather than
in intact cells (Fig. 4B).
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Fig. 4.
Structure-function analysis in murine
cholesterol 25-hydroxylase. A, expression of wild type
and mutant cholesterol 25-hydroxylase cDNAs in intact cells.
Plasmids containing no cDNA insert (lanes 1 and
4, labeled Vector), a mutant 25-hydroxylase
cDNA in which histidine codons at positions 242 and 243 were
changed to glutamine codons (lanes 2 and 5,
labeled Mutant), or a wild type 25-hydroxylase cDNA
(lanes 3 and 6, labeled Wild Type)
were introduced into CHOP cells. 24 h later, the cells were
assayed for expression of 25-hydroxylase protein by immunoblotting of
cell lysates (left panel) and for enzyme activity
in intact cells (right panel) as described under
"Experimental Procedures." B, cholesterol 25-hydroxylase
enzyme activity in cell lysates. CHOP cells were transiently
transfected with the indicated plasmids as described in A.
24 h later, cell lysates were prepared and assayed for
25-hydroxylase protein (left panel) and enzyme
activity (right panel) as described under
"Experimental Procedures." In both A and B,
mutation of the two histidine codons did not affect expression of the
protein but did inactivate the enzyme. A closely spaced doublet
corresponding to differentially glycosylated forms of the
25-hydroxylase protein (see Fig. 5A) is detected in
A, but not B, because the polyacrylamide gel in
this experiment was electrophoresed for an extended period prior to
immunoblotting.
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The subcellular localization of cholesterol 25-hydroxylase was assessed
in two ways. First, the presence and structure of asparagine-linked
carbohydrates were analyzed by endoglycosidase digestion (Fig.
5A). Expression of murine or
human cDNAs in COS cells produced two forms of the enzyme that
differed in mass by approximately 3 kDa as judged by immunoblotting
(lanes 1, 4, and 7). When total membrane proteins
from transfected cells were digested with endoglycosidase H
(lanes 2, 5 and 8) or
peptide:N-glycosidase F (lanes 3, 6, and
9), only a single form of cholesterol 25-hydroxylase was
detected that migrated with the lower molecular mass enzyme of
untreated cells. These data suggested that cholesterol 25-hydroxylase was present in the membrane fraction of the cell and that some of the
molecules were glycosylated with high mannose, asparagine-linked carbohydrates. In agreement with these results, the sequence of the
murine enzyme contains two potential sites for N-linked
glycosylation (amino acids 5 and 163, Fig. 3A), and the
human enzyme sequence contains three such sites (amino acids 5, 163, and 189, Fig. 3A).
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Fig. 5.
Analyses of asparagine-linked carbohydrates
and subcellular localization of cholesterol 25-hydroxylase.
A, simian COS cells were transfected with the indicated
cholesterol 25-hydroxylase cDNA, and membrane proteins were treated
as described under "Experimental Procedures" with endoglycosidase H
(Endo H), which cleaves high mannose and some hybrid
asparagine-linked carbohydrates or peptide:N-glycosidase F
(PNGase F), which cleaves high mannose, hybrid, and complex
asparagine-linked carbohydrates. Treated proteins were
resolved by SDS-polyacrylamide gel electrophoresis, and the expressed
cholesterol 25-hydroxylase was detected by immunoblotting with an
antipeptide antibody (U-104) that recognizes both the murine and human
proteins. B, simian COS cells were transfected with a
plasmid vector encoding a carboxyl terminus, epitope-tagged (c-Myc)
form of the murine cholesterol 25-hydroxylase. After transient
expression, cells were fixed, permeabilized, and incubated with a
murine monoclonal antibody directed against the c-Myc epitope
(green fluorescence, upper panel) and
a wheat germ agglutinin conjugate (orange fluorescence,
lower panel) that binds glycoproteins in the
Golgi compartment. A goat anti-rabbit IgG serum conjugated with
fluorescein was used to detect the primary antibody. The lectin probe
was conjugated with rhodamine. The epitope-tagged cholesterol
25-hydroxylase is detected in both the endoplasmic reticulum and Golgi
compartment of expressing cells (upper
panel).
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The second approach to examine the subcellular location of cholesterol
25-hydroxylase employed immunocytochemistry. Simian COS cells were
transfected with the plasmid pCMV-m25-COOH-myc, which encodes a
carboxyl-terminal, c-Myc epitope-tagged version of the murine
cholesterol 25-hydroxylase. After a transient expression period, cells
were permeabilized and recombinant cholesterol 25-hydroxylase detected
by indirect immunocytochemistry using a fluorescein-labeled secondary
antibody. At the same time, the transfected cells were stained with
rhodamine-labeled wheat germ agglutinin (Sigma, catalog number L5266),
a lectin that binds to glycoproteins concentrated in the Golgi
compartment. As shown in Fig. 5B, upper
panel, fluorescein signal (green) representing
cholesterol 25-hydroxylase was detected in the endoplasmic reticulum
and a perinuclear compartment of transfected cells. The perinuclear
compartment was tentatively identified as the Golgi apparatus based on
colocalization with the wheat germ agglutinin lectin (lower
panel, orange rhodamine signal). Similar results
were obtained when the c-Myc epitope was placed at the amino terminus
of the expressed murine enzyme (data not shown).
We next isolated the murine and human cholesterol 25-hydroxylase genes.
DNA sequence analysis of the isolated genomic DNAs and comparison to
the respective cDNA sequences revealed that both genes lacked
introns (Fig. 6A). This
structure was confirmed by Southern blotting analyses of murine and
human DNA and by direct amplification of the genes from genomic DNA
(data not shown). Transfection into 293 cells of a plasmid containing
the genomic DNA insert from bacteriophage clone that encompassed
~10 kb of 5'-flanking DNA and ~3 kb of 3'-flanking DNA of the
murine 25-hydroxylase gene resulted in the expression of enzyme
activity, which suggested that the isolated gene was not a pseudogene
and that requisite regulatory sequences were located close to the coding region (data not shown). The human gene was localized to chromosome 10q23.3 by somatic and radiation hybrid DNA panel mapping and fluorescence in situ hybridization (Fig. 6B).
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Fig. 6.
Comparison of murine and human cholesterol
25-hydroxylase gene sequences and chromosomal location of the human
gene. A, line up of the human and murine 25-hydroxylase
gene sequences. The MegAlign subroutine of the DNA Star sequence
analysis program was used to align the two DNA sequences for maximum
identity. The black boxes indicate identical
nucleotides. Sequences are numbered on the left.
Position 1 was arbitrarily assigned to the first nucleotides of the
illustrated sequences. The sequences specifying the initiation codons
(ATG) of both genes begin at nucleotide 11 in the 1st lines
of the DNA. The sequence specifying the termination codon of the human
gene occurs at nucleotide 827 on the 10th line. The mouse
termination codon occurs at nucleotide 905 on the 11th line.
The GenBankTM accession numbers for the murine and human
gene sequences are AF059211 and AF059212, respectively. B,
idiogram of human chromosome 10 showing location of cholesterol
25-hydroxylase gene. The position of the gene was determined by
polymerase chain reaction analyses of radiation hybrid panel DNAs and
by fluorescence in situ hybridization analyses using a
genomic DNA probe. Both methods positioned the gene on chromosome
10q23.2.
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The tissue distribution of the murine cholesterol 25-hydroxylase
mRNA was assessed by blot hybridization (Fig.
7). Low levels of a 1.5-kb mRNA were
present in the heart, lung, and kidney. The mRNA was not detected
in the livers of control mice (Fig. 7); however, it was present in RNA
from the liver of the SREBP-1a transgenic mouse used to prepare the
original cDNA expression library (data not shown). RNA blotting
experiments using commercially available filters revealed only very low
levels of human cholesterol 25-hydroxylase mRNA in 16 different
tissues (data not shown).
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Fig. 7.
Tissue distribution of murine cholesterol
25-hydroxylase mRNA. RNA blot showing cholesterol
25-hydroxylase mRNA in the heart, lung, and kidney. A commercially
obtained filter (Mouse Multiple Tissue Blot,
CLONTECH) was probed with a radiolabeled,
full-length cDNA as described under "Experimental Procedures."
After washing, the filter was exposed to x-ray film for 5 days.
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The potent regulatory effects of 25-hydroxycholesterol were first
observed in assays that measured the suppressive effects of oxysterols
on cholesterol synthesis (1, 2). To determine if the
25-hydroxycholesterol synthesized by the 25-hydroxylase enzyme could
suppress cholesterol synthesis, a line of CHO cells containing an
ecdysone-inducible 25-hydroxylase cDNA was isolated as described
under "Experimental Procedures." These cells, and a control cell
line that did not contain the 25-hydroxylase cDNA, were induced
with the ecdysone analog ponasterone, and the incorporation of
[14C]acetate into cholesterol was measured as a function
of time. As shown in Fig. 8A,
induction with ecdysone led to a marked reduction of cholesterol
synthesis in cells containing the 25-hydroxylase cDNA but had no
effect on this parameter in the control cells.
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Fig. 8.
Expression of cholesterol 25-hydroxylase
cDNA decreases cholesterol synthesis and suppresses SREBP
cleavage. A, [14C]acetate incorporation
into cholesterol was measured over a 6-h period in TR3102a cells, a
line of CHO cells transfected with an ecdysone-inducible expression
plasmid containing a murine cholesterol 25-hydroxylase cDNA ( ),
or in TR3102g cells, a line of CHO cells transfected with an
ecdysone-inducible plasmid lacking a cDNA insert ( ). Induction
with ecdysone led to a marked decrease in cholesterol synthesis only in
cells transfected with the cholesterol 25-hydroxylase cDNA.
B, Chinese hamster ovary cells (CHO-7) were transiently
transfected with the indicated plasmids as described under
"Experimental Procedures." Transfected cells were harvested, and
fractions containing membrane or nuclear proteins were prepared.
Aliquots of protein (60-80 µg) were separated by SDS-polyacrylamide
electrophoresis and blotted with antibodies directed against the
hamster SREBP-1 and -2 proteins. Antibody-antigen complexes were
visualized by enhanced chemiluminescence. An ~120-kDa protein
corresponding to uncleaved SREBP precursor was detected in the membrane
fraction of the cell (arrows on right of
upper panels), whereas an ~68-kDa cleavage product was
detected in the nuclear fraction (arrows on right
of lower panels). Expression of the transfected cholesterol
25-hydroxylase cDNA was confirmed by thin layer chromatography
assay of enzyme activity (data not shown).
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The experiments shown in Fig. 8B were carried out to
determine if expression of cholesterol 25-hydroxylase in transfected cells affected the processing of SREBP transcription factors. Cultured
CHO-7 cells were transiently transfected with either vector alone or an
expression vector containing the murine 25-hydroxylase cDNA. After
24 h, fractions enriched in membrane or nuclear proteins were
prepared from the transfected cells. Equal amounts of protein from each
subcellular compartment were separated by gel electrophoresis, and the
levels of SREBP-1 and -2 were determined by immunoblotting. Mock-transfected cells grown in the absence of sterols to induce SREBP-1 cleavage contained intact, uncleaved SREBP-1 in the membrane fraction and cleaved SREBP-1 in the nuclear fraction (Fig. 8B, lane 1). Mock-transfected cells grown in the presence of sterols (cholesterol plus 25-hydroxycholesterol) contained a majority of the
immunodetectable SREBP-1 in the membrane fraction (lane 2).
Cells transfected with the 25-hydroxylase cDNA and grown in the
absence of sterols contained a majority of SREBP-1 in the membrane
fraction even though no exogenous sterols were added (lane
3), presumably because the 25-hydroxycholesterol produced by the
expressed 25-hydroxylase suppressed cleavage of the transcription factor. Similar results were obtained when the processing of SREBP-2 was followed by subcellular fractionation and immunoblotting (Fig. 8B, right panels, lanes
4-6).
We next tested the ability of different sterols to inhibit cholesterol
25-hydroxylase (Fig. 9). In these
experiments, 293 cells were transfected with a 25-hydroxylase cDNA,
treated with 2-hydroxypropyl--cyclodextrin to remove endogenous
cholesterol, and then incubated with 3 µM
[14C]cholesterol and the indicated concentrations of
unlabeled inhibitor sterol (Fig. 9). The rank order of inhibition for
the nine sterols tested was desmosterol > cholestanol > 25-hydroxycholesterol > epicholesterol > sitosterol 
coprostanol = 25-oxo-27-nor-cholesterol (Fig. 9). When desmosterol
and [14C]cholesterol were present in equimolar amounts (3 µM), enzyme activity was decreased by 30%, whereas
coprostanol did not inhibit the enzyme at this concentration. The
observed inhibition of 25-hydroxylase activity could be due to
individual sterols acting as either true inhibitors of the enzyme
(i.e. not as substrates) or as competitors of the
cholesterol substrate. In the case of desmosterol
(5,24-cholestadien-3-ol), which cannot be 25-hydroxylated due to the
24 bond, this sterol may be acting as a true
inhibitor.
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Fig. 9.
Inhibition of cholesterol
25-hydroxylase. On Day 0 of the experiment, CHOP cells were
transiently transfected with a murine cholesterol 25-hydroxylase
expression vector using a lipofection procedure (pfx-8, Invitrogen
Corp.). On Day 1, the cells were incubated with 20 µg/ml
2-hydroxypropyl--cyclodextrin dissolved in a 1:1 mix of DMEM and F12
medium for 1.5 h to remove endogenous cholesterol. The treatment
medium was aspirated and replaced with fresh Medium D containing the
indicated sterols at different concentrations and 3.0 µM
[14C]cholesterol. On Day 2, sterols were extracted from
the media and analyzed by thin layer chromatography assay.
Autoradiograms derived from the silica gel plates are shown with the
positions of cholesterol and 25-hydroxycholesterol indicated on the
left. The percent inhibition obtained in each dish was
calculated by phosphorimage analysis and is shown below the
lanes of the chromatograms.
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DISCUSSION |
We report the isolation of cDNAs encoding enzymes that convert
cholesterol to 25-hydroxycholesterol. An expression cloning assay was
initially developed in 293 cells, and screening of >106
independent clones produced a murine cDNA specifying cholesterol 25-hydroxylase. A cDNA encoding the human homologue was
subsequently isolated by hybridization screening. The encoded murine
and human cholesterol 25-hydroxylases are hydrophobic enzymes that
synthesize 25-hydroxycholesterol, share 78% sequence identity, and are
predicted to span the membrane multiple times. In transfected cells,
the enzyme is localized to the endoplasmic reticulum and the Golgi compartment. The cholesterol 25-hydroxylase gene is expressed at low
levels in several murine tissues as judged by RNA blotting. The
expression of a cholesterol 25-hydroxylase cDNA in transfected cells suppresses the incorporation of acetate into cholesterol and the
proteolytic activation of SREBPs. Taken together, the data suggest that
cholesterol 25-hydroxylase may play an important regulatory role in
cholesterol metabolism by synthesizing 25-hydroxycholesterol, a known
sterol inhibitor of SREBP-dependent gene transcription.
Unlike other enzymes that catalyze hydroxylation reactions on sterol
and steroid substrates, the murine and human cholesterol 25-hydroxylases are not cytochrome P450s (Fig. 3A). Rather,
they belong to a growing family of enzymes that utilize oxygen and a
diiron cofactor to catalyze hydroxylation reactions on different substrates. The diiron cofactor can be either Fe
O-Fe or FeOH-Fe and is bound to the enzyme through interactions with clustered histidine or glutamate residues (37, 41). Sequence comparisons reveal
three classes of enzymes in this family, one of which is composed of
membrane-bound stearoyl-CoA desaturases, alkane hydroxylase, and xylene
monooxygenase (37). These proteins contain multiple membrane-spanning
domains and an unique arrangement of histidine clusters that are
postulated to bind the diiron cofactor and to have catalytic function.
The murine and human cholesterol 25-hydroxylases contain both of these
shared structural features (Fig. 3), and mutation of two of the
conserved histidine residues in the murine enzyme eliminates activity
(Fig. 4). Similar landmarks are also present in the C-4 sterol methyl
oxidase isolated from Saccharomyces cerevisiae and man (38,
39), a rat protein of unknown function termed neurorep 1 (42), and in
several cDNA sequences present in the data bases (e.g.
GenBankTM accession number U40941 from Caenorhabditis
elegans). The cholesterol 25-hydroxylases are 27% identical in
sequence to the C-4 sterol methyl oxidases, and they share 25-30%
sequence identity with the neurorep 1 and C. elegans
proteins. These four proteins may thus have arisen from a common
ancestor involved in sterol metabolism.
Cells transfected with the murine cholesterol 25-hydroxylase cDNA
produce authentic 25-hydroxycholesterol (Fig. 2B), and the oxysterol synthesized in cells transfected with the human cDNA comigrates with the murine product on thin layer chromatography plates
(Fig. 2A). No cDNA-dependent metabolism of
25-hydroxycholesterol, dehydroepiandrosterone, pregnenolone,
5-androstane-3, 17-diol, 5-androstane-3, 17-diol,
testosterone, dihydrotestosterone, progesterone, androsterone,
estradiol, corticosterone, or hydrocortisone was detected when these
radiolabeled compounds were added to the medium of transfected cells
(data not shown). 24-Hydroxycholesterol was converted to
24,25-dihydroxycholesterol in cells expressing a 25-hydroxylase
cDNA. These data suggest that the major enzymatic activity encoded
by the isolated cDNAs involves 25-hydroxylation of cholesterol and
24-hydroxycholesterol. However, because we have been unable to estimate
kinetic constants for the cholesterol 25-hydroxylase enzymes due to the
presence of endogenous cholesterol in the transfected cells and their
lysates, and because an exhaustive list of potential substrates has not
yet been tested, the possibility remains that the observed cholesterol
25-hydroxylase activity is a side reaction detectable only in
transfected cells overexpressing these enzymes. Additional biochemical
and genetic experiments will be performed to exclude formally this possibility.
Cellular cholesterol homeostasis is maintained by feedback repression
of genes involved in cholesterol synthesis and supply (12). Conclusive
evidence obtained in cultured cells establishes that repression is
mediated through a membrane-bound complex consisting of transcriptional
activators (SREBPs), proteases (S1P and S2P, see Ref. 26), and
associated cofactors (SCAP, see Ref. 28). When cholesterol builds up in
the cell, the activation of SREBPs by proteolysis is suppressed and
target gene transcription decreases, thus preventing a build up of
intracellular cholesterol. The active agent within the cell that
signals repression has not been identified; however, it is known that
the addition of cholesterol to cultured cells only weakly suppresses
cholesterol synthesis (43), whereas the addition of
25-hydroxycholesterol brings about a rapid decrease in both cholesterol
synthesis (1, 2) and the transcription of genes that mediate this
process (44, 45). Several indirect lines of evidence presented here
suggest that cholesterol 25-hydroxylase may be involved in the
synthesis of a co-repressor of gene transcription. First, expression of
a cholesterol 25-hydroxylase cDNA in transfected cells suppresses
cholesterol synthesis and the cleavage of endogenous SREBPs (Fig. 8).
Second, the enzyme is localized to the endoplasmic reticulum and Golgi
membranes (Fig. 5B), which are sites that would facilitate
interaction with components of the cholesterol regulatory complex.
Third, the mRNA encoding 25-hydroxylase appears to be present at
low levels in a number of tissues and cell types (Fig. 7). This
distribution would be expected of an enzyme that synthesizes a powerful
repressor of cholesterol gene expression.
How is 25-hydroxycholesterol acting as an oxysterol regulator and what
is the role of the 25-hydroxylase in its action? The regulation of
cholesterol uptake and biosynthetic enzymes shares features in common
with classic inducible and repressible systems of bacteria. On the one
hand, oxysterols, which are intermediates of cholesterol catabolism,
act as co-repressors to decrease transcription from responsive genes
just as the build up of tryptophan within a bacterial cell decreases
the synthesis of its biosynthetic enzymes (46). On the other hand,
whereas tryptophan binds to and activates a transcriptional repressor,
oxysterols act in the membrane compartment of a mammalian cell to
decrease the production of an essential positive transcription factor.
We show here that endogenously synthesized oxysterols decrease the
cleavage of SREBP precursors located in the endoplasmic reticulum;
however, the mechanism by which the sterol inhibits proteolysis remains
to be determined. Oxysterol binding is thought to induce an altered
conformation in SCAP that leads to a decrease in SREBP cleavage (12).
We are currently testing this hypothesis to explore further the role of
cholesterol 25-hydroxylase in lipid metabolism.
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ACKNOWLEDGEMENTS |
We thank Daphne Davis, Kevin Anderson, Jeff
Cormier, Mark Daris, Tammy Dinh, and Michele Laremore for excellent
technical assistance; Hitoshi Shimano and Jay Horton for SREBP-1a
transgenic mice; and Joe Goldstein and Mike Brown for critical reading
of the manuscript.
| |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grants HL20948 (to D. W. R.), the Robert A. Welch Foundation Grant I-0971 (to D. W. R.), National Institutes of Health Medical Scientist Training Grant GM08014 (to T. A. K.), the Henning and Johan
Throne-Holst Foundation for Nutrition Research (to E. G. L.), and the
Foundation BLANCEFLOR Boncompagni-Ludovisi, nee Bildt (to E. G. L.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF059213, AF059214, AF059211, and AF059212.
¶
To whom correspondence should be addressed: Dept. of Molecular
Genetics, University of Texas Southwestern Medical Center, 5323 Harry
Hines Blvd., Dallas, TX 75235-9046. E-mail:
russell{at}utsw.swmed.edu.
1
The abbreviations and trivial names used
are: cholesterol, 5-cholesten-3-ol; SREBP, sterol
regulatory element binding protein; CHOP, Chinese hamster ovary
polyoma; 25-hydroxycholesterol, cholest-5-ene-3,25-diol; 24-hydroxycholesterol, cholest-5-ene-3,24-diol;
27-hydroxycholesterol, cholest-5-ene-3,27-diol; cholestanol,
5-cholestan-3-ol; epicholesterol, 5-cholesten-3-ol;
coprostanol, 5-cholestan-3-ol; desmosterol, 5,24-cholestadien-3-ol; sitosterol, 5-cholesten-24-ethyl-3-ol; 25-oxo-27-noncholesterol, 27-nor-25-oxo-5-cholesten-3-ol; DMEM, Dulbecco's modified Eagle's medium; bp, base pair; kb, kilobase pair;
FISH, fluorescent in situ hybridization; EST, expressed sequence tag; CHO, Chinese hamster ovary.
2
The Stanford Genome Center server is available
on-line at the following address: rhserver{at}shgc.stanford.edu.
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REFERENCES |
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Abstract
Introduction
