From the Department of Biochemistry and Bioproducts
Research Center and § Institut fur Biochemische
Pharmakologie A-6020 Innsbruck, Peter Mayr-Strasse 1, Austria and
¶ Medical Research Center, Yonsei University, 134 Shinchon-dong, Sudaemoon-ku, Seoul 120-749, Korea
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ABSTRACT |
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The cDNA encoding the 471-amino
acid rat 7-dehydrocholesterol reductase (DHCR), an enzyme that has been
implicated in both cholesterol biosynthesis and developmental
abnormalities (e.g. Smith-Lemli-Opitz syndrome) in mammals,
has been cloned and sequenced, and the primary structure of the enzyme
has been deduced. The DHCR gene was mapped to chromosome 8q2.1 by
fluorescence in situ hybridization. Rat DHCR, calculated
molecular mass of 54.15-kDa polypeptide, shares a close amino acid
identity with mouse and human DHCRs (96 and 87%, respectively) as
compared with its other related proteins (e.g. fungal
sterol It has been suggested that the 19-step conversion of lanosterol to
cholesterol in mammals begins with C14-demethylation and is followed by
C14-reduction, C4-demethylation, C8 As the terminal enzyme in cholesterol biosynthesis in mammals, DHCR
(E.C. 1.3.1.21) catalyzes the reduction of the SLOS-like developmental malformations with elevation of DHC in animals
have also been observed when DHCR activity is inhibited in
vivo by either BM15.766 (16, 22, 23) or AY-9944 (24-28); these
compounds have also been shown to inhibit other cholesterogenic enzymes
including sterol 14-reductase) and exhibits high hydrophobicity
(>68%) with 9 transmembrane domains. Five putative sterol-sensing
domains were predicted to be localized in transmembrane domains 4-8,
which are highly homologous to those found in
3-hydroxymethylglutaryl-CoA reductase, sterol regulatory
element-binding protein cleavage-activating protein, and patched
protein. The polypeptide encoded by DHCR cDNA was expressed in
yeast as a 55.45-kDa myc-tagged fusion protein, which was recognized
with anti-myc monoclonal antibody 9E10 and shown to possess full DHCR
activity with respect to dependence on NADPH and sensitivity to DHCR
inhibitors. Northern blot analysis indicates that the highest
expression of DHCR mRNA was detected in liver, followed by kidney
and brain. In rat brains, the highest level of mRNA encoding DHCR
was detected in the midbrain, followed by the spinal cord and medulla.
Feeding rats 5% cholestyramine plus 0.1% lovastatin in chow resulted
in both approximately a 3-fold induction of DHCR mRNA
and a 5-fold increase of the enzymic activity in the liver.
When rats were fed 0.1% (w/w) AY-9944 (in chow) for 14-days, a
complete inhibition of DHCR activity and a significant reduction in
serum total cholesterol level were observed. However, the level of
hepatic DHCR mRNA fell only slightly, suggesting that AY-9944 may
act more rapidly at the protein level than at the level of
transcription of the DHCR gene under these conditions.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
7 isomerization, C24-reduction,
C5-desaturation, and C7-reduction (see Fig. 1) (1); this sequence has
only recently been established following the determination of the
precise location of C24-reduction (2). The enzymes involved in this
pathway (e.g. lanosterol 14
-demethylase) are major
targets for various lipid-lowering drugs, and mutations in the genes
encoding these enzymes can lead to sterol-related genetic diseases
such as Smith-Lemli-Opitz syndrome
(SLOS)1 because of mutation
in the 7-dehydrocholesterol reductase (DHCR) gene (3-5), and
desmosterolosis following mutation in the gene encoding sterol
24-reductase (6) (Fig. 1).
7 double
bond of sterol intermediates in the presence of NADPH under anaerobic
conditions (1, 7). Studies on this enzyme have recently attracted
attention because DHCR has been implicated in both cholesterol
metabolism and developmental malformations in mammals (8-10). That is,
a deficiency of DHCR activity because of genetic mutation in humans has
recently been found to cause SLOS (3-5). SLOS is an autosomal
recessive disorder with various developmental abnormalities and is
characterized by elevated 7-dehydrocholesterol (DHC) in serum body
fluids and tissues (11-19). The connection between cholesterol and
development in mammals was established based on the unexpected role of
cholesterol in the autoprocessing of sonic hedgehog protein (8-10), a
morphogen that binds to patched (PTC) protein in the developing central
nervous system and limbs. For example, mice mutated in sonic hedgehog
show a SLOS-like phenotype (20, 21).
14-reductase (29, 30) and sterol
8-isomerase (31, 32), collectively termed
AY-9944-sensitive cholesterogenic enzymes (7) (Fig.
1).
View larger version (22K):
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Fig. 1.
Sequence of the biosynthesis of cholesterol
from lanosterol (2) and some representative inhibitors blocking this
pathway. The mark denotes blocking of the reactions by
inhibitors, and the asterisk represents AY-9944-sensitive
cholesterogenic enzymes. Some of inhibitors used for this study are
indicated in bold.
The gene encoding rat DHCR had not been cloned previously. In order
that we might investigate the molecular regulation of DHCR gene
expression and the importance of impaired DHCR activity for the
development of SLOS-like phenotypes in rats (24-28), we isolated a full-length cDNA encoding rat DHCR. Work of this nature is important in order that we may understand more fully developmental cause of the SLOS phenotype and the regulation of cholesterol biosynthesis in animals.
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EXPERIMENTAL PROCEDURES |
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Materials--
A rat liver cDNA library (oligo(dT) primed
and randomly primed) and rat multiple tissue Northern blot were
purchased from CLONTECH (Palo Alto, CA). The
following materials were purchased from the manufacturers that are
indicated in parentheses: Multiple ChoiceTM Rat Brain Northern Blot
(Origen); [-32P]dCTP (3000 Ci/mmol) and Hyper x-ray
film, rainbow prestained protein marker, ECL Western blotting detection
system (Amersham Pharmacia Biotech); random-primed DNA labeling kit
(Roche Molecular Biochemicals), total RNA isolation kit (Invitrogen);
DNA preparation kit (Qiagen). Sterols included ergosterol,
7-dehydrocholesterol, lathosterol, and cholesterol (Steraloid). The
sources of the following drugs or agents are also indicated in
parentheses: AY-9944 (Wyth-Ayerst, Princeton, NJ); cholestyramine (LG
Chem, Pharmaceutical Division, Seoul, Korea);
lovastatinR (Dr. Y-K. Sim at Choongwae
Pharmaceutical Co., Suwon, Korea). Oligonucleotide primers
were synthesized by DNA synthesizer Applied Biosystem (Foster City).
Yeast strain JB811 was obtained from K. Nasmyth (Vienna, Austria).
Roswell Park Memorial Institute 1640 medium, fetal bovine serum,
penicillin/streptomycin, Dulbecco's modified Eagle's medium, and
L-glutamine were purchased from Life Technologies, Inc.
Anti-myc monoclonal antibody (9E10) was purchased from Santa Cruz
Biotechnology (Santa Cruz, CA). All other reagents were the best grade available.
Screening and Isolation of DHCR cDNA from Rat Liver
cDNA Library--
A 1.5-kb human DHCR cDNA fragment covering
the entire open reading frame (ORF) (33) was labeled with
[-32P]dCTP with a random-primed labeling kit according
to the suggested procedures for a specific activity >5 × 108 dpm/µl. The labeled fragment was denatured at
100 °C for 10 min and used as a probe to screen >3 × 106 plaques from a rat liver cDNA library according to
standard protocols provided by CLONTECH. After
tertiary screening, 12 positive clones were used for further analyses
using the slot blot hybridization (34). After digestion of 12 clones
with EcoRI, three different clones were identified; they are
7B-3-1(2.5 kb), 7B-1-1 (1.8 kb), and 7-5 (1.5 kb) (Fig.
2A), respectively.
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DNA Sequencing and Analysis of DHCR cDNA-- A DNA fragment (2.5-kb size) that was isolated by EcoRI digestion of cloned DNA from 7B-3-1 (Fig. 2A) was sequenced on both strands by the dideoxy sequencing method using a Taq Dye Deoxy Terminator Cycle sequencing kit (Applied Biosystems). Sequence and structure analyses of the cloned cDNA and its predicted protein product were carried out using BLAST programs in the GenBankTM. Rat cDNA clone homologous to human DHCR (GenBankTM accession number AF034544), Mus musculus (GenBankTM accession number AF057368), and Arabidopsis (GenBankTM accession number U49398) was analyzed with the BLASTX algorithm. Alignments and hydrophobicity analysis were calculated with the Wisconsin sequence analysis package according to the algorithms of Smith and Waterman and Kyte and Doolittle, respectively. Analysis of transmembrane segments was performed by using the following prediction programs: PHD htm at EMBL, TMpred at ISREC, SOSUI at Tokyo University, and HMMTOP prediction as described (35). Topology of rat DHCR structure was predicted by at least three of the four programs. The sequences for each protein from GenBankTM are indicated in parentheses: Chinese hamster sterol regulatory element-binding protein cleavage activation protein SCAP (280-446, GenBankTM accession number U67060); Chinese hamster 3-hydroxymethylglutaryl-CoA reductase (57-224, GenBankTM accession number L00165); mouse Niemann-Pick type C1 (617-791, GenBankTM accession number AF003348); PTC (420-589, GenBankTM accession number U46155). The alignment of these sequences with rat DHCR was performed using CLUSTALW.
Chromosomal Localization of the Rat DHCR Gene-- Two genomic EcoRI-HindIII fragments, 0.16 and 1.6 kb in length, that mainly included the coding and spacer region of 5 S rRNA, respectively, were used for fluorescence in situ hybridization analysis. The fragments were amplified by polymerase chain reaction and inserted into pUC119. The 0.16-kb fragment, which was derived from SD rats, included a 121-bp gene for mouse 5 S rRNA, which was amplified from total genomic DNA with the primers 5'-TGTCGGCGCCGCCCGCCCTC-3' and 5'-AGAGACAGAGGGGAGTCCAA-3'. One genomic DNA fragment of DHCR gene was also prepared by polymerase chain reaction using rat genomic DNA as template and the primers derived from rat genomic sequences covering the 5' end of exon 5 (5'-CATAGCAACGGGATCCA-3') and the 3' end (5'-CGTAGCCTTTCACCAAA-3') of exon 6 (2.1-kb size: 17 bp of exon 5, 2030 bp of intron, and 17 bp of exon 6) of rat gene.2 The probe was labeled by nick translation with biotin-16-dUTP (Roche Molecular Biochemicals) according to the manufacturer's protocol. Preparation of R-banded chromosomes and fluorescence in situ hybridization were performed as described (36).
Heterologous Expression of Myc-tagged Rat DHCR in Saccharomyces cerevisiae-- Rat DHCR cDNA was subcloned into c-myc-tagged yeast episomal plasmid c-myc-YEp351ADC1, and transformation of S. cerevisiae JB811 (ade2-1 leu2-3, 112 pep4-3 trp1-289 ura3-52) was performed as described (33). Cells were harvested at an A600 of 1.2 and lysed with glass beads in 50 mM Tris-HCl (pH 7.4), 1 mM EDTA, 0.1 mM phenylmethylsulfonylfluoride. For the preparation of microsomes, the lysates were spun for 5 min at 500 × g (4 °C), and the supernatant was pelleted for 45 min at 100,000 × g (4 °C) as described (33). Immunoblotting with a 9E10 c-myc monoclonal antibody following SDS/polyacrylamide gel electrophoretic analysis was exactly the same as described previously (33). Protein concentrations were determined by the method of Bradford (37) using bovine serum albumin as a standard.
Animals, Diet Feeding, and Drug Treatment-- Male Sprague-Dawley rats (200-250-g body weight) were maintained on a standard rodent chow under a reverse light cycle (light 6:00 p.m-6:00 a.m.; dark 6:00 a.m.-6:00 p.m.) as described previously (30). In the diet feeding experiments, each diet-fed group (4 to 5 rats/group) was fasted for 12 h and then fed diets supplemented with various agents such as 5% cholestyramine plus 0.1% lovastatin (CL-diet) in or 0.1% AY-9944 in chow for 14 days (30). At the end of the treatment, the rats were fasted for 24 h and killed by CO2 inhalation. Blood was drawn by heart puncture and analyzed for serum lipids. The plasma serum cholesterol was determined by an automatic analyzer Hitachi 7150. Low density lipoprotein cholesterol was calculated from the method of Friedewald formulation (38).
Preparation of Microsomes and Enzyme Assays--
Rats that had
been fed under various diet conditions were killed by decapitation at
the midpoint of the dark period (10 p.m. for rats), and their livers
were excised and processed for microsome preparation as described
previously (30). A standard assay for DHCR was carried out using 300 µM substrate (ergosterol, 7-dehydrocholesterol, or
lathosterol) that had been suspended in the detergent Tween 80 (70:1,
detergent:sterol) and 1 to 2 mg of microsomal protein as described (7).
For the in vitro inhibition experiments for both rat liver
microsomes and yeast overexpressed protein, drugs or agents were
dissolved in dimethyl sulfoxide (Me2SO) such that the final
concentration of Me2SO was less than 0.3% (w/v) that of
the incubation mixture (2). The sterol concentration in enzyme assay samples was measured in a Hewlett Packard gas
chromatograph 5890 II or a Young-In GC520 gas chromatograph (Seoul,
Korea) (FID, capillary column; SAC-5, 5% diphenyl, 95%
dimethylsiloxane, 30 m × 0.25 mm, 0.25 µm inner diameter, flow
rate 2.44 ml/min) using 5-cholestane as a standard (30, 32).
RNA Preparation and Northern Blot Analysis--
For the effects
of diet on DHCR gene expression, total RNA from liver of either
CL-diet, AY-9944-fed, or normal diet-fed rats were prepared with total
RNA isolation kit (Invitrogen) according to the manufacturer's
procedures. Fifteen µg of each tissue RNA were separated on a 1.2%
(w/v) agarose gel in 30% (v/v) formaldehyde and blotted. RNAs were
probed with 32P-labeled rat DHCR cDNA (GenBankTM
accession number AF071500). A rat -actin cDNA probe was used as
a control. After hybridization (42 °C, 50% (v/v) formamide), blots
were washed at low stringency (e.g. 0.2 × SSC (1 × SSC = 0.15 M NaCl and 0.015 M sodium
citrate), 0.1% (w/v) SDS at 22 °C). Rat multiple tissue Northern
blot (CLONTECH) and Multiple ChoiceTM Rat Brain
Northern Blot (Origen) were probed with [32P]-labeled rat
DHCR cDNA. The blots were exposed to Hyper x-ray film with
intensifying screens at
70 °C for longer than 48 h. The bands
that appeared in the blots were quantitatively analyzed with a
Molecular Dynamics 425DE PhosphorImager using ImageQuaNT program. DHCR
mRNA signals obtained from different tissues by the Northern blot
hybridization were normalized to those of
-actin mRNA expression.
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RESULTS AND DISCUSSION |
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Cloning and Sequence Analysis of Rat DHCR cDNA-- Screening of a rat liver cDNA library (CLONTECH) with the human cDNA probe yielded three positive clones (i.e. 7B-3-1, 7B-1-1, and 7-5) (Fig. 2A) of approximately >3.0 × 106 plaques screened. The nucleotide sequences of these clones were determined on both strands and revealed that all of these clones (e.g. clone 7B-3-1) encode the rat DHCR cDNA (Fig. 2B). The DHCR cDNA (clone 7B-3-1) is 2356 nucleotides long and consists of a 48-nucleotide 5'-untranslated region followed by a 1416-nucleotide coding region and a 892-nucleotide 3'-untranslated region. As expected, the nucleotide sequence of rat DHCR showed highest homology with mouse DHCR. That is, the coding region (ORF) of the cDNA sequence (1416 nucleotides) showed 96, 87, and 36% identity with mouse (GenBankTM accession number AF057368), human (GenBankTM accession number AF034544), and Arabidopsis thaliana (GenBankTM accession number U49398) DHCR cDNA sequences, respectively. Unlike human DHCR cDNA, which has an additional polyadenylation site, rat DHCR cDNA contains only a single polyadenylation site, similar to the mouse ortholog (3). A striking feature of the rat DHCR mRNA sequence is that it contains an exceptionally long 3'-untranslated region (892 nucleotides), indicating the polyadenylation site is quite distant from the termination codon. The deduced amino acid sequence of rat DHCR (Fig. 2B) predicts a 471-residue protein with a calculated molecular mass of 54,155 Da, quite similar to the human (475 amino acid (33)) and mouse (471 amino acids (3)) enzymes.
Structural Feature of Rat DHCR Protein--
Fig.
3A presents the alignment of
rat DHCR polypeptide with other mammalian enzymes (human and mouse (3,
33)) and A. thaliana (39). At the polypeptide level, the rat
DHCR ORF shows 96 and 87% identity to the mouse and human DHCR
polypeptides over 471 amino acids (Table
I). The relatively high degree of
sequence similarity in ORF between DHCRs of different species suggests that they may be involved in fundamental physiological processes that are evolutionarily conserved between animals and plants. There is
also a significant degree of similarity between the amino acid sequence
of DHCR and related proteins (Table I). In particular, a relatively
high similarity is seen between DHCR and fungal sterol 14-reductase (47%) (39), human lamin B receptor (48%),
which has been suggested as a putative sterol
14-reductase (41), and fungal sterol
24-reductase (43%) (42). However, although rat sterol
8-isomerase is a member of AY-9944-sensitive
cholesterogenic enzymes, its homology to rat DHCR is low (29%) (Table
I).
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Several structural motifs are conserved among the mouse, human, and rat proteins (Fig. 3A). They are hydrophobic regions, transmembrane domain (marked as box), and glycosylation sites (dots). The DHCR polypeptides contains a highly conserved motif, GVQEGA (residue 124-129), which is thought to represent potential N-myristoylation sites (43, 44). The deduced amino acid sequence of DHCR also contains a total of 23 serine (4.8%) and 22 threonine residues (4.7%) located throughout the backbone that may serve as putative phosphorylation sites for serine/threonine kinases as suggested (45). There are also three putative protein kinase C phosphorylation sites (i.e. 73-75 (TGR), 89-91 (TAK), and 437-439 (THR)) (46, 47) and two tyrosine kinase phosphorylation sites (i.e. 372-378 (KAIECSY) and 450-458 (KYGRDWERY)) (48, 49). The deduced amino acid sequence of rat DHCR contains four potential N-linked glycosylation sites (Asn-Xaa-(Ser/Thr) (50-52) at 8-11 (NASK), 248-251 (NLSF), 283-286 (NETW), and 403-406 (NYTG) (Fig. 3A). The first and the fourth (at 8-11 and 403-406) are positioned in relatively long loops of the DHCR that are projected into the ER lumen. The second and third are localized inside the transmembrane domain (248-251) or very close to it (283-286).
A hydropathy plot indicates that there is a large proportion (>68%)
of hydrophobic amino acids throughout the polypeptide backbone (except
for the region of amino acid residues 3-30 and 349-388) of the
deduced amino acid sequence (Fig. 3B). More than 68% of the
rat DHCR polypeptide appears to be hydrophobic, a feature shared by
other cholesterogenic microsomal enzymes (Fig. 3B)
(e.g. 3-hydroxymethylglutaryl-CoA reductase (53)). The
existence of at least nine transmembrane -helical domains is
predicted by Kyte-Doolittle hydrophobicity analysis (54), in keeping
with our previous observation that solubilization of membrane-bound DHCR by various nonionic detergents (e.g. CHAPS) was
extremely difficult to accomplish (7).
The most intriguing feature of DHCR protein structure is that it
contains a sterol-sensing domain (SSD) (Fig.
4A), which has been well
reported in five other sterol-mediating regulatory proteins: sterol
regulatory element-binding protein (55), sterol regulatory element-binding protein cleavage activation protein (56, 57), PTC (58),
TRC8, a PTC-related protein (35), and Niemann Pick-type C1 protein
(59); the SSD of rat DHCR was predicted to be localized at
transmembrane segments 4-8, covering amino acid residues 177 and 358 (Fig. 4A). The alignment of the SSDs of various proteins is
shown in Fig. 4B. The putative SSD region of rat DHCR
(residues 177-358) showed 47% similarity and 12.6% identity to the
SSD of mouse sterol regulatory element-binding protein cleavage
activation protein (280-446). The significance of the similarity of
rat DHCR to other sterol-related proteins is currently unknown.
However, among human SLOS patients, three missense mutations have been found in this putative SSD region: A247V, V326L, and R352W (3). These
mutations in human DHCR also resulted in 95-97% reduction in enzyme
activity (3), suggesting that loss both of enzyme activity and
sterol-sensing response may contribute to development of SLOS during
embryogenesis.
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Assignment of the Rat DHCR Gene to Chromosome 8q2--
DNA
isolated from the rat genomic library by polymerase chain reaction was
hybridized to the region of the long arm of rat chromosome 8q2 from 31 metaphase chromosome slides of 4 Sprague-Dawley rats (Fig.
5). Using replicative R-banding, we
confirmed that the DHCR genome probe was localized to rat chromosome
8q2.1 (data not shown). Human and mouse DHCR genes were previously
localized to chromosome 11q12-13 (5) and 7F5 (3), respectively.
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Functional Analysis of the Cloned DHCR cDNA--
To examine
the function of cloned rat DHCR cDNA, a control expression vector
for the cDNA (i.e. c-myc-YEp351ADC1) encoding a
myc-tagged DHCR protein (myc-DHCR) was transfected into JB811 S. cerevisiae cells (33). Because it is already known that the expression of a myc-tagged DHCR significantly increased DHCR activity in yeast crude microsomes (33), we designed and used a myc-DHCR expression vector for the experiment. An advantage of this tagged protein is that it enables the use of an immunoblot detection assay
system without the need for a DHCR-specific antibody, which is not
available at the present time. As anticipated, transfected yeast cells
expressed an approximately 55.45-kDa rat DHCR protein, which was
recognized with the anti-myc monoclonal antibody 9E10 (Fig.
6A). No protein was detected
in the immunoblot of yeast cells transfected with control vector.
Immunoblotting of expression of myc-DHCR showed excellent agreement in
calculated molecular mass of DHCR (i.e.
Mr 54,550) in 12% SDS/polyacrylamide gels. Rat
DHCR activity in yeast crude membranes is illustrated in Fig. 6B. The overexpressed cloned myc-DHCR fusion protein showed
a similar pattern of inhibition to that previously seen in the rat liver microsomal protein upon exposure to AY-9944 (IC50 = 0.243 µM) or U18666A (IC50 = 2.690 µM) when enzyme activities were determined under
conditions where the activities were linear with respect to time and
substrate concentration (Fig. 6C). This observation is in
excellent agreement with our previous results that had been obtained
with rat hepatic DHCR in which IC50 of AY-9944, for
example, was estimated to be 0.25 µM (7). The
overexpressed protein was not inhibited significantly by tamoxifen (a
specific inhibitor of sterol 8-isomerase) (60) or
miconazole (a specific inhibitor of lanosterol 14
-demethylase) (61,
62). Furthermore, the DHCR overexpressed in yeast exhibited essentially
the same properties as those of hepatic microsomes (Table
II) (7). For example, the overexpressed DHCR was highly sensitive to the presence of AY-9944 (Fig.
6C) and sulfhydryl-binding agents and dependent on NADPH
(i.e. NADH cannot substitute for NADPH as a proton donor)
(Table II). The activity of the overexpressed DHCR protein was mildly
affected by the presence of oxygen and phosphate ions but not by some
metal ions (4 mM), as previously seen in the case of rat
hepatic DHCR (7). Thus, these results suggest that the cloned rat
hepatic DHCR cDNA indeed encodes functional DHCR protein, and there
is essentially no difference in the functionality of the enzyme when it
is expressed in yeast.
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Tissue Distribution of DHCR mRNA--
The distribution of
mRNAs encoding DHCR in different rat tissues was examined by
Northern blot analysis using a rat DHCR cDNA ORF probe. The signals
of DHCR mRNA on blots were normalized to those of -actin
mRNA. As shown in Fig. 7A,
the liver (100% A = 41 by PhosphorImager analysis)
exhibited the highest signal, and that is followed by the kidney (13%
A = 5.3) and the brain (9% A = 3.7).
No signal was detected in other rat tissues (heart, testis, lung,
spleen, and skeletal muscle). This result suggests that the liver is
the major site of rat DHCR mRNA expression. To determine tissue
distribution of DHCR mRNA in the rat brain, Multiple Choice
Northern brain blot (Origen) was hybridized with rat cDNA probe in
which the hybridization signal of each part of brain was normalized to
that of rat
-actin. As shown in Fig. 7B, midbrain(as
100%) showed the highest densities followed by spinal cord and
medulla. It should be noted that there is a marked difference between
human (33) and rat DHCR mRNA distribution. That is, the mRNA
for rat DHCR is expressed highly in rat liver and moderately in brain
and kidney, which corresponds well to the known tissue distribution of
the enzyme activity,2 whereas ubiquitous expression of
human DHCR mRNA (in organs such as the adrenal gland, liver, brain,
heart, and testis) is seen (33). In particular, in humans the level of
DHCR mRNA expression in the brain is very high, at almost the same
as that of the liver (33), whereas in the rat the level of mRNA of
liver is predominant over that of brain (almost 14-fold).
The significance of this difference between the two species remains to
be determined.
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In Vivo Regulation of the Hepatic DHCR Gene by Lipid-lowering
Drugs--
To determine whether DHCR mRNA levels are regulated by
feeding lipid-lowering drugs (e.g. cholestyramine,
lovastatin, and AY-9944), DHCR mRNAs in liver tissue from chow-fed
and CL-diet-fed rats (30, 7) were quantified by Northern blot analysis
(Fig. 8). In the CL-diet-fed rats, both
hepatic DHCR mRNA (expressed as an optical density of the
hybridized bands in the blot by densitometric scanning) and the
specific enzymic activity (expressed as nmol/min/mg of protein) were
increased nearly 3-fold (i.e. in optical densities of
mRNA signals, 35.8 ± 6 (control) versus 97.3 ± 8 (CL-diet), A values normalized to -actin signal
n = 3) and 5-fold (i.e. in enzymic
activities, 4.42 ± 0.7 (control) versus 21.37 ± 2.8 (CL-diet), n = 3), respectively, relative to the
control rats in three independent experiments (Fig. 8). Thus, the DHCR
induction by CL-diet shown here and previously seen (7) was mainly
because of elevation of DHCR mRNA levels in these animals. Although
AY-9944 has long been known to inhibit DHCR activity, the molecular
nature of this inhibitor has not been reported. This inhibitor in fact also causes teratogenic SLOS-like phenotypes in rodents (24, 26, 28).
Northern blot analysis was performed on RNA from livers of rats fed
AY-9944 for 14 days. Surprisingly, the level of DHCR mRNA was not
much changed in liver at this dose of AY-9944 (0.1%, w/w,
approximately 7.0 mg/kg/day). However, there was a huge reduction in
serum lipids (e.g. average values of 65% total cholesterol,
49% low density lipoprotein cholesterol, and 38% triglyceride of
control (n = 3)
(30)3 and complete inhibition
of enzyme activity (to an undetectable level) in vivo. The
failure of the same dose of AY-9944 to alter the level of expression of
DHCR mRNA (from 35.8 to 30.1; A values normalized to
-actin signal) suggests that the mechanism of action of this
inhibitor may occur most rapidly at the protein level, as seen
previously (7), than at the transcriptional level. However, we do not
rule out the possibility that in the longer term (e.g. >1
month) ingestion of higher doses of AY-9944 (e.g. >75 mg/kg
(27)), which have been known to cause SLOS-like phenotype in rats, may
modulate the level of transcription of the DHCR gene. This point
remains to be clarified.
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In conclusion, our results on the molecular characterization of DHCR
cDNA may provide many useful opportunities with regard to 1) the
elucidation of the molecular regulatory mechanism for DHCR gene
expression in vitro and in vivo, 2) the creation
of an animal model for investigation and treatment of SLOS, 3)
investigation of structure-function relationships in DHCR, and 4) other
useful pharmacokinetic studies including determination of the effect of
targeted mutations during investigation of treatment strategies. In
particular, the elucidation of the role of sterol-sensing domains in
DHCR expression may provide some clues about the connection between
this enzyme and developmental malformations such as the SLOS-like
phenotypes in mammals.
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ACKNOWLEDGEMENT |
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We thank Drs. H. Glossmann, F. Moebius, and D. J. Adams for their help and critical readings of this manuscript. We also thank Dr. Yoischi Matsuda for his help on fluorescence in situ hybridization work and Dr. W.-T. Lee and J.-H. Kim for their assistance on this project.
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FOOTNOTES |
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* This work was supported by grants from the Korean Ministry of Science and Technology Molecular Medicine Project 98MM-0204A03 (to Y.-K. P.) and Korea Science and Engineering Foundation through the Bioproducts Research Center at Yonsei University (9514-0401-00-12-3 (to Y.-K. P.)).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) AF071500.
To whom correspondence should be addressed: Yonsei University,
Dept. of Biochemistry, 134 Shinchon-dong, Sudaemoon-ku, Seoul, 120-749, Korea. Tel.: 82-2-361-2702; Fax: 82-2-362-9897; E-mail: paikyk{at}bubble.yonsei.ac.kr.
2 J. N. Lee and Paik, Y.-K., unpublished data.
3 S-H. Bae and Y.-K. Paik, unpublished observation.
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ABBREVIATIONS |
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The abbreviations used are:
SLOS, Smith-Lemli-Opitz syndrome;
DHCR, 7-dehydrocholesterol reductase;
kb, kilobase(s);
ORF, open reading frame;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
SSD, sterol-sensing domain;
AY-9944, trans1,4-bis(2-chlorobenzylaminomethyl)cyclohexane
dihydrochloride;
BM15. 766, 4(2-(1-(4-chlorocinnamyl)-piperazin-4-ylethyl)-benzoic acid;
CL-diet, 5% cholestyramine plus 0.1% lovastatin in normal chow;
IC50, a concentration of inhibitor required for 50%
inhibition;
lanosterol, 4,4',14-trimethyl-5
-cholesta-8,24-dien-3
-ol;
lathosterol, 5
-cholesta-7-en-3
-ol;
PTC, patched protein;
tamoxifen, trans-2-[4-(1,2-diphenyl-1-butenyl)phenoxy]-N,N-dimethylethylamine;
U18666A, 3-
-[2-(diethylamino)ethoxy]androst-5-en-17-one.
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