Disruption of the gene encoding 3b-hydroxysterol
D
14
-reductase (Tm7sf2) in mice does not impair
cholesterol biosynthesis
Anna M. Bennati
1
, Gianluca Schiavoni
1
, Sebastian Franken
2
, Danilo Piobbico
3
, Maria A. Della
Fazia
3
, Donatella Caruso
4
, Emma De Fabiani
4
, Laura Benedetti
5
, Maria G. Cusella De Angelis
5
,
Volkmar Gieselmann
2
, Giuseppe Servillo
3
, Tommaso Beccari
1
and Rita Roberti
1
1 Department of Internal Medicine, University of Perugia, Italy
2 Institut fu
¨r Physiologische Chemie, Rheinische Friedrich-Wilhelms-Universita
¨t, Bonn, Germany
3 Department of Clinical and Experimental Medicine, University of Perugia, Italy
4 Department of Pharmacological Sciences, University of Milan, Italy
5 Department of Experimental Medicine, University of Pavia, Italy
In cholesterol biosynthesis, lanosterol undergoes
removal of the methyl group at C14, leading to the
formation of C14–C15 unsaturated sterol intermedi-
ates. The enzymatic activity responsible for the reduc-
tion of the introduced double-bond, 3b-hydroxysterol
D
14
-reductase (EC 1.3.1.70), is carried out by the
endoplasmic reticulum (ER) protein delta14-sterol
reductase (C14SR) encoded by the TM7SF2 gene
Keywords
3beta-hydroxysterol delta14-reductase;
cholesterol biosynthesis; gene expression;
lamin B receptor; Tm7sf2
Correspondence
R. Roberti, Department of Internal Medicine,
Laboratory of Biochemistry, University of
Perugia, Via del Giochetto, 06122 Perugia,
Italy
Fax: +39 075 585 7428
Tel: +39 075 585 7426
E-mail: roberti@unipg.it
(Received 24 May 2008, accepted 11
August 2008)
doi:10.1111/j.1742-4658.2008.06637.x
Tm7sf2 gene encodes 3b-hydroxysterol D
14
-reductase (C14SR, DHCR14),
an endoplasmic reticulum enzyme acting on D
14
-unsaturated sterol interme-
diates during the conversion of lanosterol to cholesterol. The C-terminal
domain of lamin B receptor, a protein of the inner nuclear membrane
mainly involved in heterochromatin organization, also possesses sterol
D
14
-reductase activity. The subcellular localization suggests a primary role
of C14SR in cholesterol biosynthesis. To investigate the role of C14SR and
lamin B receptor as 3b-hydroxysterol D
14
-reductases, Tm7sf2 knockout
mice were generated and their biochemical characterization was performed.
No Tm7sf2 mRNA was detected in the liver of knockout mice. Neither
C14SR protein nor 3b-hydroxysterol D
14
-reductase activity were detectable
in liver microsomes of Tm7sf2
()⁄))
mice, confirming the effectiveness of
gene inactivation. C14SR protein and its enzymatic activity were about half
of control levels in the liver of heterozygous mice. Normal cholesterol
levels in liver membranes and in plasma indicated that, despite the lack of
C14SR, Tm7sf2
()⁄))
mice are able to perform cholesterol biosynthesis.
Lamin B receptor 3b-hydroxysterol D
14
-reductase activity determined in
liver nuclei showed comparable values in wild-type and knockout mice.
These results suggest that lamin B receptor, although residing in nuclear
membranes, may contribute to cholesterol biosynthesis in Tm7sf2
()⁄))
mice. Affymetrix microarray analysis of gene expression revealed that
several genes involved in cell-cycle progression are downregulated in the
liver of Tm7sf2
()⁄))
mice, whereas genes involved in xenobiotic metabolism
are upregulated.
Abbreviations
C14SR ⁄DHCR14, 3b-hydroxysterol D
14
-reductase; C27D
8
,5a-cholesta-8(9)-en-3b-ol; C27D
8,14
,5a-cholesta-8(9),14-dien-3b-ol; C29D
8
,
4,4-dimethyl-5a-cholesta-8(9)-en-3b-ol; C29D
8,14
, 4,4-dimethyl-5a-cholesta-8(9),14-dien-3b-ol; ER, endoplasmic reticulum; HEM, Hydrops-
Ectopic calcification-Moth-eaten skeletal dysplasia; LBR, lamin B receptor.
5034 FEBS Journal 275 (2008) 5034–5047 ª2008 The Authors Journal compilation ª2008 FEBS
[1,2]. A second protein of the inner nuclear membrane,
the lamin B receptor (LBR), catalyzes the 3b-hydroxys-
terol D
14
-reductase reaction, as demonstrated by its
ability to complement C14SR-deficient yeast strains
[3,4] and by enzymatic assay of the protein overexpres-
sed in transfected COS-1 cells [5]. Recently, the mouse
gene encoding 3b-hydroxysterol D
14
-reductase has been
termed Dhcr14 [6]; in this study the former gene name
Tm7sf2 will be used. Tm7sf2 is located on chromosome
19A.
The expression of cholesterol biosynthesis genes is
regulated by cell sterol levels through the action of the
transcription factor SREBP-2 [7,8]. In HepG2 hepa-
toma cells, sterol starvation results in induction of the
TM7SF2 gene, C14SR protein and 3b-hydroxysterol
D
14
-reductase activity. In addition, human TM7SF2
promoter is regulated by SREBP-2 [5]. Therefore, the
adaptability of TM7SF2 gene to the needs of choles-
terol biosynthesis appears well established. On the con-
trary, LBR gene expression is not responsive to sterol
starvation conditions [5] and its importance in choles-
terol biosynthesis remains unravelled.
The lack of 3b-hydroxysterol D
14
-reductase caused
by mutations of the LBR gene was previously indi-
cated as responsible for Hydrops-Ectopic calcification-
Moth-eaten skeletal dysplasia (HEM or Greenberg
dysplasia) [9–11]. The severe phenotype of natural
Lbr
()⁄))
mutants, ichthyosis mice, has been described,
but no information on cholesterol levels and ⁄or sterol
intermediate accumulation has been reported [12].
Therefore, inactivation of Tm7sf2 gene in mice would
provide insights into the role of both genes encoding
3b-hydroxysterol D
14
-reductase, Tm7sf2 and Lbr,in
cholesterol biosynthesis.
While Tm7sf2
()⁄))
mice generated in our laboratory
were under characterization, a paper was published
describing mice defective for Lbr (Lbr
()⁄))
, ichthyosis
mice), defective for Dhcr14 ⁄Tm7sf2 (Dhcr14
(D4-7 ⁄D4-7)
)
or defective for Dhcr14 ⁄Tm7sf2 and heterozygous for
Lbr (Lbr
(+ ⁄))
:Dhcr14
(D4-7 ⁄D4-7)
) [6]. The paper states
that HEM dysplasia is a laminopathy not caused by
3b-hydroxysterol D
14
-reductase deficiency. Mutants
have distinct physical and biochemical phenotypes, but
no sterol abnormalities were detected in liver, whereas
marked elevations of D
14
-sterols were seen in brain of
Lbr
(+ ⁄))
:Dhcr14
(D4-7 ⁄D4-7)
mice.
Here, we describe the generation of Tm7sf2
()⁄))
mice and their biochemical characterization. Determi-
nation of Tm7sf2 and Lbr mRNA expression in differ-
ent mouse tissues, expression of C14SR and LBR
proteins in liver and a measure of their 3b-hydroxys-
terol D
14
-reductase activity are reported. Despite the
lack of C14SR, Tm7sf2
()⁄))
mice are apparently
healthy and have normal cholesterol levels in liver
membranes and in plasma, suggesting that LBR can
function as 3b-hydroxysterol D
14
-reductase in vivo.
Furthermore, microarray analysis of gene expression in
liver comparing wild-type and Tm7sf2
()⁄))
mice has
been performed.
Results
Identification of mouse Tm7sf2 gene
Genomic clones of Tm7sf2 were isolated by screening
a mouse 129 ⁄SvJ genomic library, subcloned and
sequenced. Comparison of the mouse genomic and the
cDNA sequences revealed that the exon–intron organi-
zation of the mouse Tm7sf2 gene is highly similar to
the homologous human gene [1]. The gene spans
5 kb and consists of 10 exons and 9 introns. Table 1
shows the size of the exons and introns and the
sequence of the exon–intron junctions. All 5¢donor
and 3¢acceptor splice sites conformed to the consensus
Table 1. Exon–intron organization of mouse Tm7sf2 gene. Exon and intron length is reported in parentheses.
Exon Intron
Sequence of exon–intron junction
5¢splice donor–3¢splice acceptor
1 (143 bp) 1 (349 bp) GGGCCGTTGG gtaaatggga–––ctctttccag GCGTCGCGGC
2 (197 bp) 2 (100 bp) CTGCACGAAG gtgtgtgatc–––gtacccgcag GTGGCCGAAG
3 (55 bp) 3 (274 bp) CCTATTAATG gtgactgggg–––tgtggttcag GCTTCCAGGC
4 (195 bp) 4 (84 bp) GGAAACTCAG gtgagaaggg–––ttgttcccag GAAATTCCAT
5 (104 bp) 5 (2112 bp) CATTGGCTGG gtatgctgac–––acttctttag GTTTTCATTA
6 (120 bp) 6 (88 bp) CTGGTATGAG gtgagactgg–––gttcctgcag GAGTCTGTCC
7 (169 bp) 7 (214 bp) CTCCTTAAGG gtcagtagga–––cttccctcag TTATTGGTTA
8 (81 bp) 8 (80 bp) AGCGTGGCTG gtaagctggg–––gtatttctag GTCTTGAGAC
9 (123 bp) 9 (250 bp) TTGCCCTGTG gtgagtgggt–––ttccctccag GGCTATCCCA
10 (253 bp) CTATCCCATC–––
A. M. Bennati et al. Tm7sf2 knockout mice
FEBS Journal 275 (2008) 5034–5047 ª2008 The Authors Journal compilation ª2008 FEBS 5035
GT–AG rule. The transcription initiation site, deter-
mined by RACE, was located at )91 bp upstream the
ATG start codon. A polyadenylation signal
(AATAAA) is present 49 bp downstream of the stop
codon. The genomic sequence has been submitted to
GenBank under accession number EU672836.
Tm7sf2 and Lbr expression in mouse tissues
Tm7sf2 and Lbr relative mRNA expression was mea-
sured in adrenal, brain, heart, kidney, liver, lung,
ovary and testis of 8-week-old mice using qRT-PCR.
The highest Tm7sf2 mRNA abundance was found in
liver, followed by ovary, testis, kidney and brain
(Fig. 1A). Testis and lung showed the highest Lbr gene
expression, followed by heart, ovary, kidney and liver
(Fig. 1B).
Tm7sf2 versus Lbr expression was determined by
using Lbr as the internal calibrator for each tissue.
Table 2 shows comparable expression of the two genes
in ovary, kidney and adrenal gland. Compared with
Lbr,8- and 2.5-fold higher Tm7sf2 expression was
found in liver and brain, respectively. By contrast, Lbr
versus Tm7sf2 expression was 5-, 12- and 16-fold
higher in testis, heart and lung, respectively.
Generation of Tm7sf2-null mice
Tm7sf2 gene was disrupted in HM1 mouse embryonic
stem cells using a targeting vector in which exon 5 was
interrupted by a neomycin resistance cassette (Fig. 2B).
The homologous recombination between the targeting
vector and the endogenous Tm7sf2 allele resulted in
insertion of the neomycin phosphotransferase gene into
exon 5. Of 96 ES cell clones surviving G418 selection,
four targeted clones were identified by PCR analysis
and by Southern blot of EcoRI-digested genomic
DNA and two of them were injected into blastocysts.
Only clone E-53 generated germline transmitting chi-
meric male founders, which were intercrossed with
C57 ⁄B6 females to generate outbred strains. Offspring
were genotyped by PCR, which produced the expected
230 and 388 bp fragments from wild-type and dis-
rupted allele, respectively (Fig. 2B,C). Genotyping was
confirmed by Southern blot of EcoRI-digested DNA.
Labelled 8.9 and 7.8 kb fragments were obtained from
wild-type and mutated allele, respectively (Fig. 2B,D).
Offspring from heterozygous intercrosses showed the
typical Mendelian distribution of wild-type, heterozy-
gous and homozygous.
Tm7sf2
()⁄))
mice develop normally, appear healthy
and are fertile. Histopathological analysis of liver, kid-
ney, adrenal and brain did not reveal differences
between control and mutant mice (data not shown).
Followed over a 3-month period, Tm7sf2
()⁄))
mice
grow at the same rate as littermate control mice.
Groups of control and Tm7sf2
()⁄))
female weighed at
14 months of age were 27.7 ± 1.9 g (n= 9) and
30.9 ± 1.9 g (n= 7), respectively. No apparent age-
dependent problems were observed in females or males
over a 14-month period. These results confirm previ-
ously reported data [6].
0.0
0.2
0.4
0.6
0.8
1.0
1.2
A
B
Tm7sf2 relative mRNA expression
Adrenal
Brain
Heart
Kidney
Liver
Lung
Ovary
Testis
Adrenal
Brain
Heart
Kidney
Liver
Lung
Ovary
Testis
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Lbr relative mRNA expression
Fig. 1. Expression of Tm7sf2 and Lbr mRNA in mouse tissues.
RNA was extracted from pooled tissues of three 8-week-old male
mice (females were used for ovary). Relative mRNA expression
was determined by qRT-PCR using liver and testis as internal cali-
brators for Tm7sf2 (A) and Lbr (B) genes, respectively. Experiments
were performed in triplicate and repeated twice with different RNA
preparations. Reported data are mean ± SD.
Tm7sf2 knockout mice A. M. Bennati et al.
5036 FEBS Journal 275 (2008) 5034–5047 ª2008 The Authors Journal compilation ª2008 FEBS
To test whether the mutation abolishes the expres-
sion of C14SR, RNA was extracted from liver and the
cDNA was synthesized by RT-PCR using the forward
and reverse primers that amplify the entire cDNA.
Figure 3A shows that the cDNA is absent in
Tm7sf2
()⁄))
mice, whereas the expected 1.3 kb frag-
ment is obtained in control mice. Although no quanti-
tative PCR was performed, the cDNA was about half
of the control in heterozygous mice. Gapdh was ampli-
fied in parallel as housekeeping gene.
Western blot analysis of microsomes prepared from
liver was performed using anti-(bovine C14SR) serum,
which cross-reacts with the mouse protein. Figure 3B
shows that C14SR protein is absent in Tm7sf2
()⁄))
mice, whereas the band intensity is about half of
control (0.55 ± 0.09) in heterozygous mice, thus con-
firming the results obtained with the cDNA.
To investigate whether disruption of the Tm7sf2
gene modifies Lbr mRNA expression, qRT-PCR was
performed in tissues of 8-week-old wild-type and
Tm7sf2
()⁄))
mice, using the wild-type as internal cali-
brator for each tissue. No significant differences of Lbr
mRNA expression in Tm7sf2
()⁄))
mice, compared
with that of wild-type mice, were found in the exam-
ined tissues (adrenal, brain, heart, kidney, liver, lung,
ovary and testis) (data not shown).
Western blot analysis of nuclear membranes from
liver confirmed that LBR is expressed at about the
same level of control in Tm7sf2
()⁄))
mice (Fig. 3C).
A
B
C
D
Fig. 2. Structure and targeted disruption of
Tm7sf2 gene. (A) The conversion of 4,4-
dimethyl-5a-cholesta-8(9),14-dien-3b-ol
(C29D
8,14
) to 4,4-dimethyl-5a-cholesta-8(9)-
en-3b-ol (C29D
8
) is catalysed by C14SR and
LBR. (B) Tm7sf2 genomic structure, target-
ing vector, and disrupted allele. Exon (filled
box) and intron lengths are approximately to
scale. PCR primers a, b, and neo are indi-
cated by arrows. The 3¢-probe used in
Southern blot experiments spans exon 10.
The size of EcoRI-digested wild-type and
disrupted allele is reported. (C) PCR geno-
typing of heterozygous Tm7sf2 intercross.
Primers a and b (B) amplify a 230-bp frag-
ment from the wild-type allele. Primers a
and neo (B) amplify a 388-bp fragment from
the disrupted allele. (D) Southern blot analy-
sis of mouse tail DNA isolated from the
progeny of a mating between heterozygous
parents. DNAs were digested with EcoRI
and hybridized with the 3¢-probe indicated in
(B).
Table 2. Tm7sf2 and Lbr expression in mouse tissues. Total RNA
was extracted from tissues of 8-week-old mice and retrotranscribed
as reported in Experimental procedures. Tm7sf2 and Lbr mRNA
expression was measured by qRT-PCR using the specific primers
(see Experimental procedures) and Lbr as internal calibrator for
each tissue. Hprt was used as the reference gene for sample nor-
malization. Data are mean ± SD of two experiments performed in
triplicate.
Tissue Tm7sf2 ⁄Lbr ratio
Adrenal 0.68 ± 0.06
Brain 2.40 ± 0.51
Heart 0.09 ± 0.03
Kidney 0.90 ± 0.09
Liver 7.95 ± 0.71
Lung 0.06 ± 0.01
Ovary 1.06 ± 0.13
Testis 0.19 ± 0.05
A. M. Bennati et al. Tm7sf2 knockout mice
FEBS Journal 275 (2008) 5034–5047 ª2008 The Authors Journal compilation ª2008 FEBS 5037
3b-Hydroxysterol D
14
-reductase activity
3b-Hydroxysterol D
14
-reductase activity in microsomes
or in intact nuclei prepared from liver of 6-week-old
mice was determined in vitro by incubating the enzyme
source in the presence of the C27D
8,14
sterol substrate.
Activity was measured on the basis of C27D
8
forma-
tion and C27D
8,14
disappearance, evaluated as the peak
area ratio between the individual sterol and cholestane,
the internal standard.
No enzymatic activity was detected in liver micro-
somes of Tm7sf2
()⁄))
mice, whereas reduced activity
was detected in heterozygous compared with wild-type
mice (Fig. 4). The data are in accordance with C14SR
expression detected by western blot. The enzymatic
activity measured in intact nuclei, which can be
referred as LBR activity, was comparable in
Tm7sf2
()⁄))
and Tm7sf2
(+ ⁄+)
mice (Fig. 4).
The contribution of C14SR and LBR to the 3b-hy-
droxysterol D
14
-reductase reaction in liver from wild-
type mice can be evaluated. On the basis of the
amount of incubated proteins (see legend in Fig. 4) in
the experimental conditions used, C14SR-specific activ-
ity was approximately eightfold higher than that of
LBR. Because 4.4 and 1.5 mg proteinÆg
)1
of fresh tis-
sue were recovered as microsomes and nuclei, respec-
tively, C14SR enzymatic activity is >20-fold higher
than LBR enzymatic activity. This result is in accor-
dance with the high Tm7sf2 mRNA expression in liver,
compared with Lbr (Table 2).
Sterol determinations
Cholesterol concentration in liver microsomal mem-
branes of 6-week-old mice was measured both by
GC-MS analysis and by densitometry analysis of mem-
brane lipids separated by TLC. Despite the lack of
C14SR activity in microsomes, normal cholesterol
levels were found in these membranes (Table 3). No
differences were found between male and female mice.
Cholesterol biosynthetic precursors, including
C29D
8,14
, were not detectable by GC-MS analysis
of microsomal sterols, indicating that C29D
8,14
inter-
mediate was not accumulated in Tm7sf2
()⁄))
mice.
kb +/+ +/– –/–
Gapdh
1.5
Tm7sf2 cDNAABC
kDa +/+ –/–
66
45
31
+/–
Anti-C14SR
Microsomes
66
kDa +/+ –/–
Anti-LBR
Nuclear membranes
Fig. 3. C14SR and LBR expression in liver. (A) PCR analysis of Tm7sf2 mRNA in the liver of Tm7sf2
()⁄))
, heterozygous and wild-type mice.
The primers used (see Experimental procedures) amplify a 1285-bp fragment. (B) Western blot analysis of liver microsomes. The anti-(bovine
C14SR) serum recognises a protein band with an apparent molecular mass of 40 kDa. Equal amounts of protein (30 lg) were loaded in
each lane and checked by Ponceau staining of poly(vinylidene difluoride) membranes after protein transfer (data not shown). (C) Western
blot analysis of liver nuclear membranes. The anti-(human LBR) serum recognizes a protein band with an apparent molecular mass of
66 kDa. Equal amounts of proteins (50 lg) were loaded in each lane and checked by Ponceau staining of poly(vinylidene difluoride) mem-
branes after protein transfer (data not shown). Experiments were repeated at least three times with different RNA (A) or membrane (B and
C) preparations. One representative experiment is reported.
0.0
0.5
1.0
1.5
2.0
2.5
Sterol/cholestane (peak area ratio)
Nuclei
t0+/+ +/– –/– +/+ –/–
Microsomes
Fig. 4. 3b-Hydroxysterol D
14
-reductase activity. Microsomes
(0.25 mg protein) and intact nuclei (0.5 mg protein) prepared from
liver of wild-type, heterozygous and Tm7sf2
()⁄))
mice were
assayed for 3b-hydroxysterol D
14
-reductase activity by incubation
for 30 min with C27D
8,14
in the conditions described in Experimen-
tal procedures. Enzymatic activity was evaluated on the basis of
the decrease of peak area ratio between m⁄z426 and m⁄z372
ions (C27D
8,14
⁄5a-cholestane, filled columns) and the increase of
peak area ratio between m⁄z428 and m⁄z372 ions (C27D
8
⁄5a-
cholestane, open columns) at the expected retention time. C27D
8
was absent at zero incubation time both in the microsomes and in
nuclei. Data shown are mean ± SD (n= 3).
Tm7sf2 knockout mice A. M. Bennati et al.
5038 FEBS Journal 275 (2008) 5034–5047 ª2008 The Authors Journal compilation ª2008 FEBS