Disruption of the gene encoding 3β-hydroxysterol Δ14-reductase (Tm7sf2) in mice does not impair cholesterol biosynthesis

Disruption of the gene encoding 3b-hydroxysterolD14-reductase (Tm7sf2) in mice does not impaircholesterol biosynthesisAnna M. Bennati1, Gianluca Schiavoni1, Sebastian Franken2, Danilo Piobbico3, Maria A. DellaFazia3, Donatella Caruso4, Emma De Fabiani4, Laura Benedetti5, Maria G. Cusella De Angelis5,Volkmar Gieselmann2, Giuseppe Servillo3, Tommaso Beccari1 and Rita Roberti1

1 Department of Internal Medicine, University of Perugia, Italy

2 Institut fur Physiologische Chemie, Rheinische Friedrich-Wilhelms-Universitat, 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-hydroxysterolD14-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: [email protected]

(Received 24 May 2008, accepted 11

August 2008)

doi:10.1111/j.1742-4658.2008.06637.x

Tm7sf2 gene encodes 3b-hydroxysterol D14-reductase (C14SR, DHCR14),

an endoplasmic reticulum enzyme acting on D14-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

D14-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 D14-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 D14-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 D14-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 D14-reductase; C27D8, 5a-cholesta-8(9)-en-3b-ol; C27D8,14, 5a-cholesta-8(9),14-dien-3b-ol; C29D8,

4,4-dimethyl-5a-cholesta-8(9)-en-3b-ol; C29D8,14, 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 ª 2008 The Authors Journal compilation ª 2008 FEBS

[1,2]. A second protein of the inner nuclear membrane,

the lamin B receptor (LBR), catalyzes the 3b-hydroxys-terol D14-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 D14-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-hydroxysterolD14-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 D14-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 D14-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 D14-reductase deficiency. Mutants

have distinct physical and biochemical phenotypes, but

no sterol abnormalities were detected in liver, whereas

marked elevations of D14-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 D14-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 D14-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 ª 2008 The Authors Journal compilation ª 2008 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

1.2A

B

Tm

7sf2

rel

ativ

e m

RN

A e

xpre

ssio

n

Adrenal

Brain

Heart

Kidney

LiverLung

Ovary

Testis

Adrenal

Brain

Heart

Kidney

LiverLung

Ovary

Testis

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Lb

r re

lati

ve m

RN

A e

xpre

ssio

n

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.


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

(C29D8,14) to 4,4-dimethyl-5a-cholesta-8(9)-

en-3b-ol (C29D8) 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



3b-Hydroxysterol D14-reductase activity

3b-Hydroxysterol D14-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 C27D8,14 sterol substrate.

Activity was measured on the basis of C27D8 forma-

tion and C27D8,14 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 D14-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

C29D8,14, were not detectable by GC-MS analysis

of microsomal sterols, indicating that C29D8,14 inter-

mediate was not accumulated in Tm7sf2() ⁄ )) mice.

kb +/+ +/– –/–

Gapdh

1.5

Tm7sf2 cDNAA B CkDa +/+ –/–

66

45

31

+/–

Anti-C14SR

Microsomes

66kDa +/+ –/–

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

Ste

rol/c

ho

lest

ane

(pea

k ar

ea r

atio

)

Nucleit0 +/+ +/– –/– +/+ –/–

Microsomes

Fig. 4. 3b-Hydroxysterol D14-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 D14-reductase activity by incubation

for 30 min with C27D8,14 in the conditions described in Experimen-

tal procedures. Enzymatic activity was evaluated on the basis of

the decrease of peak area ratio between m ⁄ z 426 and m ⁄ z 372

ions (C27D8,14 ⁄ 5a-cholestane, filled columns) and the increase of

peak area ratio between m ⁄ z 428 and m ⁄ z 372 ions (C27D8 ⁄ 5a-

cholestane, open columns) at the expected retention time. C27D8

was absent at zero incubation time both in the microsomes and in

nuclei. Data shown are mean ± SD (n = 3).



Plasma cholesterol was comparable between 6-week-

old Tm7sf2(+ ⁄ +) and Tm7sf2() ⁄ )) mice, both in males

and females (Table 3). Groups of 14-month-old female

mice were also analysed, and comparable plasma

cholesterol levels were seen in wild-type and knockout

mice (Table 3).

Affymetrix oligonucleotide array hybridization

and data analysis

The expression of 45 101 transcripts represented on

the Affymetrix murine GeneChipMOE430 2.0 was

quantified in liver from wild-type and Tm7sf2() ⁄ ))

mice. Duplicate hybridizations were performed for

each sample, using the wild-type condition as a control

to measure fold change of gene expression in knockout

mice. Expression values were calculated using the

Robust Multi-Array Analysis (RMA) plugin [13] of the

genespring 7.3 software (Agilent Technologies, Milan,

Italy). Using this analysis, 1119 transcripts with a

SD < 0.2 were selected in the comparison knock-

out ⁄wild-type mice. Volcano Plot analysis of this tran-

script list was performed to identify transcripts with a

defined minimal fold change and statistically significant

P-value for a t-test of differences between samples. By

selecting a fold change > 1.5 and a P-value < 0.01, 66

transcripts were identified as increased (Table 4) and 41

as decreased (Table 5) in livers of Tm7sf2() ⁄ )) mice.

Table 4 shows that several transcripts of oxidoreduc-

tases are increased, including members of cyt p450

families. Glutathione S-transferase, involved in xenobi-

otic metabolism, is also increased. Some genes involved

in cell proliferation and cell-cycle progression show

decreased transcripts (Table 5). qRT-PCR analysis was

applied to some of the genes that show the highest

up- or down expression comparing knockout to the

wild-type mice. Although with different fold changes,

the results obtained in the microarray experiment were

confirmed (Tables 4 and 5).

The complete panel of genes upstream and down-

stream Tm7sf2 in the cholesterol biosynthetic pathway

and Lbr were analysed carefully using less stringent

parameters (P-value for the t-test < 0.05). Neverthe-

less, no difference in their expression was found in the

liver of Tm7sf2() ⁄ )) mice.

Discussion

The discovery that inborn errors in cholesterol biosyn-

thesis are the cause of human malformation syndromes

characterized by severe developmental abnormalities

[14] highlights the role of cholesterol in mammalian

embryonic development. Indeed, cholesterol is a cova-

lent ligand of hedgehog morphogenic signalling pro-

teins [15]. Normal cholesterol biosynthesis is required

for cell membrane assembly and for the biosynthesis of

different biologically active sterol molecules. Further-

more, the cholesterol biosynthetic pathway furnishes a

variety of intermediate molecules involved in several

cell processes. In light of this, it is predictable that

mouse models bearing inactivation of any of the genes

of cholesterol biosynthesis are characterized by

impaired cell functions and developmental abnormali-

ties incompatible with life. Indeed, several mutant mice

lacking genes of the pre- or post-squalene segment of

the cholesterol biosynthetic pathway, that are lethal to

various degrees, have been described [16–23].

The mutant mouse model generated in our labora-

tory by inactivation of the Tm7sf2 gene lacks the ER

3b-hydroxysterol D14-reductase, C14SR. Nevertheless,

mice are viable and do not exhibit a pathological phe-

notype, as previously reported for Dhcr14(D4-7 ⁄ D4-7)

mice [6]. Using the antibody raised in our laboratory

against bovine liver C14SR, we were able to verify a

reduction and the complete absence of C14SR expres-

sion in liver microsomes of Tm7sf2(+ ⁄ )) and

Tm7sf2() ⁄ )) mice, respectively. In accordance with

these data, no 3b-hydroxysterol D14-reductase activity

is detectable in liver microsomes of Tm7sf2() ⁄ )) mice.

Despite the lack of 3b-hydroxysterol D14-reductase

activity of C14SR, normal cholesterol biosynthesis

occurs in Tm7sf2() ⁄ )) mice. Indeed, the level of

Table 3. Cholesterol in plasma and liver microsomal membranes. Total plasma cholesterol was measured using a commercial kit. For micro-

somal membrane cholesterol determination, samples were saponified and cholesterol was measured by GC-MS and by densitometric analy-

sis of lipids separated by TLC and stained as described in Experimental procedures.

Plasma cholesterol (mgÆdL)1)

Microsomal membrane cholesterol (nmolÆmg)1

protein)

Tm7sf2(+ ⁄ +) Tm7sf2() ⁄ )) Tm7sf2(+ ⁄ +) Tm7sf2() ⁄ ))

Male (6 weeks) 82.2 ± 10.3 (n = 8) 77.6 ± 14.5 (n = 12) 73.9 ± 5.8 (n = 6) 71.5 ± 6.5 (n = 6)

Female (6 weeks) 70.7 ± 12.9 (n = 15) 66.0 ± 11.8 (n = 10)

Female (14 months) 65.6 ± 21.1 (n = 9) 81.7 ± 20.2 (n = 7)



Table 4. Genes whose transcripts increase in liver of Tm7sf2() ⁄ )) mice. Total RNA from the pooled livers of three 6-week-old mice was pre-

pared for Affymetrix oligonucleotide hybridization as described in Experimental procedures. The relative mRNA expression of each gene in

livers of knockout mice compared with wild-type control is shown. Parameters for Volcano plot statistical analysis were fold change > 1.5

and P-value for the t-test < 0.01. Results from duplicate hybridizations are listed by fold change.

Affymetrix

probeset

identification GenBank

Gene

symbol Gene name

Fold

change

1438211_s_at NM_016974 Dbp D-site albumin promoter

binding protein

6.21 ⁄ 4.05a

1448793_a_at NM_011521 Sdc4 syndecan 4 3.43 ⁄ 4.80a

1420603_s_at NM_009016, NM_009017,

NM_009018, NM_020030,

NM_198193, XM_001006217

Raet1a, Raet1b,

Raet1c, Raet1d,

Raet1e

retinoic acid early transcript

1alpha, beta, gamma, delta, 1E

3.18

1449347_a_at NM_001081642, NM_021365,

NM_183094, XM_001471704,

XM_001471888, XM_001475552,

XM_001487778, XM_978371,

XR_035676, XR_035679

LOC100044048,

LOC100044049,

LOC100046087,

Xlr4a, Xlr4b,

Xlr4c, Xlr4e

X-linked lymphocyte-regulated

4A, 4B, 4C, 4E

3.02

1444438_at XM_356089, XM_904518 Cib3 calcium and integrin binding

family member 3

2.98 ⁄ 1.66a

1424853_s_at NM_010011, NM_201640,

XM_001471913

Cyp4a10, Cyp4a31,

LOC100044218

cytochrome P450, family 4,

subfamily a, polypeptides 10&31

2.68 ⁄ 1.83a

1416318_at NM_025429 Serpinb1a serine (or cysteine) peptidase

inhibitor, clade B, member 1a

2.65

1444810_at – – – 2.55

1421363_at NM_010003 Cyp2c39 cytochrome P450, family 2,

subfamily c, polypeptide 39

2.53 ⁄ 17.2a

1439560_x_at XM_488763, XM_489668 EG432995 predicted gene, EG432995 2.49

1419700_a_at NM_008935 Prom1 prominin 1 2.38

1453345_at NM_001081205 Npal1 NIPA-like domain containing 1 2.33

1421040_a_at NM_008182 Gsta2 glutathione S-transferase,

alpha 2 (Yc2)

2.32

1435459_at NM_018881 Fmo2 flavin containing monooxygenase 2 2.26

1435893_at NM_013703 Vldlr very low density lipoprotein

receptor

2.14

1423875_at NM_145505, XM_001473083 AI450540,

LOC100044843

expressed sequence AI450540 2.13

1431240_at NM_053165 Clec2h C-type lectin domain family 2,

member h

2.13

1421041_s_at NM_008181, NM_008182,

XM_001478795

Gsta1, Gsta2,

LOC100042295

glutathione S-transferase, alpha 1

(Ya); glutathione S-transferase,

alpha 2 (Yc2)

2.06

1438194_at – 2900019G14Rik RIKEN cDNA 2900019G14 gene 2.06

1456973_at – – – 2.05

1452501_at NM_010002 Cyp2c38 cytochrome P450, family 2,

subfamily c, polypeptide 38

1.98

1422903_at NM_010745 Ly86 lymphocyte antigen 86 1.93

1415932_x_at NM_015731 Atp9a ATPase, class II, type 9A 1.92

1418213_at NM_033373 Krt23 keratin 23 1.92

1444706_at – Nav2 neuron navigator 2 1.91

1423627_at NM_008706 Nqo1 NAD(P)H dehydrogenase, quinone 1 1.88

1450505_a_at NM_001034851, NM_025459 1810015C04Rik RIKEN cDNA 1810015C04 gene 1.85

1450648_s_at NM_207105 H2-Ab1 histocompatibility 2, class II

antigen A, beta1

1.84

1417900_a_at NM_013703 Vldlr very low density lipoprotein receptor 1.83

1455316_x_at XM_915804 ENSMUSG00000073624 predicted gene, ENSMUSG00000073624 1.81

1458585_at – – – 1.80

1447643_x_at NM_011415 Snai2 snail homolog 2 (Drosophila) 1.78



cholesterol in microsomes from liver, as well as plasma

cholesterol, were comparable between wild-type and

Tm7sf2() ⁄ )) mice. Plasma cholesterol was still normal

in a group of 14-month-old female mice.

In addition to C14SR, the inner nuclear membrane

protein LBR exhibits 3b-hydroxysterol D14-reductase

activity [5]. Reciprocal increase of Lbr and Tm7sf2 gene

expression was detected in the liver of 1-day-old

Dhcr14(D4-7 ⁄ D4-7) and Lbr() ⁄ )) mice, respectively [6].

These results, together with the absence of D14-sterols

accumulation, supported the hypothesis that C14SR and

LBR provide redundancy with respect to 3b-hydroxy-sterol D14-reductase activity [6]. We analysed Lbr

expression in several adult mice tissues, including liver,

Table 4. Continued

Affymetrix

probeset

identification GenBank

Gene

symbol Gene name

Fold

change

1427604_a_at NM_015731 Atp9a ATPase, class II, type 9A 1.78

1439293_at NM_153584 BC031353 cDNA sequence BC031353 1.76

1419430_at NM_007811 Cyp26a1 cytochrome P450, family 26, subfamily a,

polypeptide 1

1.75

1450884_at NM_007643 Cd36 CD36 antigen 1.74

1420879_a_at NM_018753 Ywhab tyrosine 3-monooxygenase ⁄ tryptophan

5-monooxygenase activation protein,

beta polypeptide

1.73

1429831_at NM_031376 Pik3ap1 phosphoinositide-3-kinase adaptor protein 1 1.72

1418710_at NM_007652 Cd59a CD59a antigen 1.71

1448978_at NM_019867 Ngef neuronal guanine nucleotide exchange factor 1.68

1446731_at – A730016A17 Fanconi anemia, complementation group F 1.68

1417025_at NM_010382 H2-Eb1 histocompatibility 2, class II antigen E beta 1.68

1422975_at NM_008604 Mme membrane metallo endopeptidase 1.63

AFFX-r2-Bs-

thr-M_s_at

– – – 1.63

1417629_at NM_011172 Prodh proline dehydrogenase 1.63

1417017_at NM_007809 Cyp17a1 cytochrome P450, family 17, subfamily

a, polypeptide 1

1.63

AFFX-ThrX-M_at – – – 1.62

1431916_at NM_001012306 Hsd3b3 hydroxy-delta-5-steroid dehydrogenase,

3 beta- and steroid delta-isomerase 3

1.62

1417828_at NM_007474 Aqp8 aquaporin 8 1.61

1448595_a_at NM_009052 Bex1 brain expressed gene 1 1.60

1428083_at NR_003513, XR_035481,

XR_035482

2310043N10Rik RIKEN cDNA 2310043N10 gene 1.60

1448568_a_at NM_015747 Slc20a1 solute carrier family 20, member 1 1.60

1420549_at NM_010259 Gbp1 guanylate nucleotide binding protein 1 1.59

1453109_at NM_029847 Arsk arylsulfatase K 1.59

1416193_at NM_001083957, NM_009799 Car1 carbonic anhydrase 1 1.58

1415822_at NM_009128 Scd2 stearoyl-Coenzyme A desaturase 2 1.58

1445862_at NM_001081154 4921513D23Rik RIKEN cDNA 4921513D23 gene 1.58

1424683_at NM_001034851, NM_025459 1810015C04Rik RIKEN cDNA 1810015C04 gene 1.57

1429104_at NM_172397 Limd2 LIM domain containing 2 1.57

1449067_at NM_031197 Slc2a2 solute carrier family 2 (facilitated glucose

transporter), member 2

1.53

1422479_at NM_019811 Acss2 acyl-CoA synthetase short-chain family

member 2

1.52

1443056_at – – – 1.52

1442418_at – B930096F20Rik RIKEN cDNA B930096F20 gene 1.51

1457760_at – A930004J17Rik RIKEN cDNA A930004J17 gene 1.50

1456225_x_at NM_175093 Trib3 tribbles homolog 3 (Drosophila) 1.50

1437176_at NM_001033207 AI451557 expressed sequence AI451557 1.50

a Fold change determined by qRT-PCR.



Table 5. Genes whose transcripts decrease in liver of Tm7sf2() ⁄ )) mice. See legend to Table 4.

Affymetrix

probeset

identification GenBank Gene symbol Gene name Fold change

1444297_at NR_002861, XM_001471933 LOC100044164,

Serpina4-ps1

serine (or cysteine) peptidase inhibitor,

clade A, member 4, pseudogene 1

)5.26

1444296_a_at NR_002861, XM_001471933 LOC100044164

Serpina4-ps1



)4.05

1448092_x_at NR_002861, XM_001471933 LOC100044164

Serpina4-ps1



)4.02

1427797_s_at NM_007799 Ctse cathepsin E )3.92-

1416664_at NM_023223 Cdc20b cell division cycle 20 homolog (S. cerevisiae) )3.60 ⁄ )10.8a

1424638_at NM_007669 Cdkn1ab cyclin-dependent kinase inhibitor 1A (P21) )3.38 ⁄ )8.9a

1417764_at NM_025965, XM_911969 LOC636537, Ssr1 signal sequence receptor, alpha )3.30

1420451_at NM_021370 Accn5 amiloride-sensitive cation channel 5,

intestinal

)3.23 ⁄ )4.1a

1421447_at NM_008262, XM_001480325 LOC100048479,

Onecut1 (Hnf6)bone cut domain, family member 1 )3.02 ⁄ )6.1a

1424278_a_at NM_001012273, NM_009689 Birc5 (survivin40)b baculoviral IAP repeat-containing 5 )2.98

1450252_at NM_008262 Onecut1 (Hnf6)b one cut domain, family member 1 )2.40

1425948_a_at NM_026232 Slc25a30 solute carrier family 25, member 30 )2.35

1452754_at NM_029720 Creld2 cysteine-rich with EGF-like domains 2 )2.29

1456974_at NM_008262 Onecut1 (Hnf6)b one cut domain, family member 1 )2.27

1420836_at NM_026232 Slc25a30 solute carrier family 25, member 30 )2.20

1425127_at NM_153193 Hsd3b2 hydroxy-delta-5-steroid dehydrogenase,

3 beta- and steroid delta-isomerase2

)2.17

1416076_at NM_172301, XM_001005050,

XM_485921, XM_900988,

XR_030797, XR_033681

Ccnb1b, Ccnb1-rs1,

EG434175,

LOC667005

cyclin B1; cyclin B1, related sequence 1;

predicted gene, EG434175

)2.13

1422001_at NM_010565 Inhbc inhibin beta-C )2.10

1423397_at NM_133894 Ugt2b38 UDP glucuronosyltransferase 2 family,

polypeptide B38

)2.08

1419669_at NM_011178 Prtn3 proteinase 3 )1.98

1417370_at NM_011575 Tff3 trefoil factor 3, intestinal )1.96

1424695_at NM_025912 2010011I20Rik RIKEN cDNA 2010011I20 gene )1.93

1442051_at NM_013549 Hist2h2aa1 histone cluster 2, H2aa1 )1.92

1437073_x_at – AV025504 expressed sequence AV025504 )1.89

1450440_at NM_010279 Gfra1 glial cell line derived neurotrophic

factor family receptor alpha 1

)1.84

1424118_a_at NM_025565 Spc25b SPC25, NDC80 kinetochore complex

component, homolog (S. cerevisiae)

)1.83

1416299_at NM_011369 Shcbp1b Shc SH2-domain binding protein 1 )1.82

1433955_at NM_145125 Brwd1b bromodomain and WD repeat domain

containing 1

)1.77

1419319_at NM_011316 Saa4 serum amyloid A 4 )1.77

1448314_at NM_007659 Cdc2ab cell division cycle 2 homolog A (S. pombe) )1.76

1425993_a_at NM_013559 Hsp110 heat shock 105kDa ⁄ 110kDa protein 1 )1.76

1416757_at NM_026507 Zwilchb zwilch, kinetochore associated, homolog

(Drosophila)

)1.75

1424684_at NM_024456 Rab5c RAB5C, member RAS oncogene family )1.73

1417991_at NM_007860 Dio1 deiodinase, iodothyronine, type I )1.67

1425282_at NM_146042 Ibrdc2b ring finger protein 144B )1.67

1455892_x_at – – – )1.67

1425107_a_at NM_013584 Lifrb leukemia inhibitory factor receptor )1.66

1449824_at XM_355243 Prg4 proteoglycan 4 (megakaryocyte stimulating

factor, articular superficial zone protein)

)1.66

1448756_at NM_009114 S100a9 S100 calcium binding protein A9 (calgranulin B) )1.66

1429379_at NM_053247 Lyve1 lymphatic vessel endothelial hyaluronan receptor 1 )1.64

1439695_a_at NM_183046 Mphosph1b kinesin family member 20B )1.64

a Fold change determined by qRT-PCR. b Genes involved in cell proliferation and cell-cycle progression.



and we did not find significant differences between wild-

type and Tm7sf2() ⁄ )) mice. In the liver, the result

obtained by qRT-PCR was further confirmed by the

Affymetrix oligonucleotide array hybridization experi-

ment. In addition, the expression of LBR protein in

nuclei from liver and its enzymatic activity were compa-

rable in wild-type and Tm7sf2() ⁄ )) mice. These results

indicate that, at least in adult Tm7sf2() ⁄ )) mice, LBR

can account for normal cholesterol biosynthesis without

increasing its expression. The discrepancy in Lbr gene

expression between 1-day-old Dhcr14(D4-7 ⁄ D4-7) and

adult Tm7sf2() ⁄ )) mice could be related to higher cho-

lesterol biosynthetic activity during development, com-

pared with adult mice. In vitro, C14SR exhibits higher

cholesterol biosynthetic capacity than LBR. Although it

cannot be excluded that this is due to the experimental

conditions used for enzymatic activity determination

(substrate and ⁄or cofactors), this result is in accordance

with higher Tm7sf2 gene expression in liver, compared

with Lbr. In vivo, the regulation of Tm7sf2 gene expres-

sion by cholesterol levels could represent a mechanism

to adapt 3b-hydroxysterol D14-reductase activity to

increased cholesterol needs.

The post-squalene segment of cholesterol biosynthe-

sis takes place in the ER. Among the enzymes involved

in this pathway, LBR is the unique residing in the

inner nuclear membranes. The N-terminal nucleoplas-

mic domain of LBR is involved in heterochromatin

organization [24], whereas the transmembrane domain

is responsible for 3b-hydroxysterol D14-reductase activ-

ity. Following its synthesis, LBR diffuses laterally from

the ER throughout the nuclear pore membranes and it

is retained in the inner nuclear membranes by its bind-

ing to the lamina [25]. It has been speculated whether

the ability of LBR to synthesize cholesterol in vivo is

restricted to its presence in the ER or in the nuclear

envelope [26]. Deficiency of LBR during granulopoiesis

results in hypolobulation of the mature neutrophil

nucleus, suggesting that LBR helps make the extra

nuclear membranes required during the nuclear lobula-

tion of granulopiesis [27]. Although LBR was not

detectable in liver microsomes, it cannot be excluded

that in vivo LBR supports cholesterol biosynthesis

when present in the ER. Alternatively, the mobility of

sterol intermediates in the lateral plane of the mem-

brane to reach enzymes differently compartmentalized

should occur.

The role of cholesterol and intermediates of its bio-

synthesis in cell growth and division is well known

[28,29]. The stringency of the requirement for choles-

terol during proliferation and cell-cycle progression

was investigated in promyelocytic HL-60 cells by com-

parison with other sterols of the biosynthetic pathway.

In the absence of exogenous cholesterol, accumulation

of intermediate sterols upstream 7-dehydrocholesterol,

including C29D8,14, resulted in the inhibition of cell

proliferation and cell cycle arrest in G2 ⁄M phase [30].

Affymetrix oligonucleotide array analysis showed that

several genes involved in cell proliferation and cell-

cycle progression have decreased expression in the liver

of Tm7sf2() ⁄ )) mice. Although no altered phenotype

has been observed in Tm7sf2() ⁄ )) mice so far, we

could speculate that an impaired response of liver cells

to proliferative stress is conceivable in these mice.

The evaluation of C14SR and LBR expression and

the determination of their enzymatic activity in the

liver of wild-type and Tm7sf2() ⁄ )) mice reinforce the

hypothesis that LBR and C14SR provide enzymatic

redundancy with respect to cholesterol synthesis [6].

Indeed, no sterol abnormalities were detected in

Tm7sf2() ⁄ )) mice. The significance of this enzymatic

redundancy is not clear. Even though it can be hypoth-

esized that multiple mechanisms have been developed

during evolution to ensure cholesterol biosynthesis, it

should be considered that the 3b-hydroxysterol D14-

reductase reaction is the only one in the post-squalene

pathway to be catalysed by two different enzymes. It is

worth noting that Drosophila LBR lacks sterol reduc-

tase activity, which could have been lost during evolu-

tion [31]. Different roles for Tm7sf2 and Lbr genes

during development or in tissues can be expected, rais-

ing the question of how 3b-hydroxysterol D14-reductase

activity can be switched between C14SR and LBR and

which regulatory mechanisms are involved.

Experimental procedures

Materials

Cholesterol, 5a-cholestane, and commercial antibodies were

purchased from Sigma (Milan, Italy). The polyclonal anti-

(bovine C14SR) serum was raised in our laboratory as

previously described [2]. The polyclonal anti-(human LBR)

serum was a kind gift of H. Hermann (German Cancer

Research Center, Heidelberg, Germany) [32]. Complete pro-

tease inhibitor cocktail tablets were from Roche Diagnos-

tics (Milan, Italy). RNAlater RNA Stabilization Reagent,

Qiazol Lysis Reagent, and RNeasy Mini Kit were from

Qiagen (Milan, Italy). QuickChange Site-Directed Muta-

genesis Kit, AffinityScript Multiple Temperature Reverse

Transcriptase, and Brilliant� SYBR� Green QPCR Master

Mix were purchased from Stratagene (La Jolla, CA, USA).

RiboLock RNase inhibitor, random hexamer primers, Taq

DNA polymerase, and restriction enzymes were from

Fermentas (St Leon-Rot, Germany). 5a-Cholesta-8,14-dien-3b-ol was synthesized as previously described [2].



Identification of mouse Tm7sf2 gene

Mouse Tm7sf2 mRNA was identified by NCBI database

comparison with the human mRNA (accession no.

AF096304). The mouse cDNA was synthesized by RT-PCR

using liver RNA as template and the following primers: for-

ward 5¢-ATGTCGACGATCATGACTTCTCGTGAGG-3¢and reverse 5¢-ATGTCGACTTCAACCTCTTAGGTG

GACC-3¢ (the SalI restriction site introduced for subclon-

ing in pBlueScript vector is given in italics). The sequenced

1285 bp cDNA (accession no. AF480070) encodes a puta-

tive protein 86% identical to human C14SR [1,2].

The cDNA was used to screen a 129 ⁄ SvJ mouse genomic

library in Lambda FIX II vector (Stratagene). Five library

clones that contained different chromosomal DNA frag-

ments were isolated and two contained the entire gene, as

demonstrated by Southern blot hybridization analysis.

Restriction endonuclease fragments from the Tm7sf2 geno-

mic clones were subcloned into pBlueScript vector and

sequenced.

Generation of Tm7sf2 knockout mice

A Tm7sf2 targeting vector was generated by subcloning

into pBluescript vector a 5380 bp KpnI ⁄XbaI fragment,

containing exons 1–5 of the gene (Fig. 2B). The neomycin

phosphotransferase gene (neo) was inserted in a SalI

restriction site generated by in vitro mutagenesis in

exon 5. The vector was linearized with NotI and electro-

porated into HM1 mouse embryonic stem cells (strain

129Sv ⁄ ola) [33]. After selection of G418-resistant embry-

onic stem cell colonies, homologous recombination was

established by PCR using a primer internal to the neo

gene (5¢-AGAACCTGCGTGCAATCCATCTTG-3¢) and a

3¢-primer external to the targeting construct (5¢-AAGCT

CTGCCTCCTGCATCAGC-3¢), which produced a 2560-

bp fragment. PCR cycling conditions were: 15 min

denaturation at 95 �C followed by 45 cycles of 30 s at

95 �C, 45 s at 64 �C, 3 min at 72 �C, and a final exten-

sion of 10 min at 72 �C. The positive clones were

subjected to Southern blotting after EcoRI genomic DNA

digestion, using a 450-bp probe located to the 3¢-end of

the gene and external to the targeting construct (Fig. 2B).

The probe detects an 8.9 kb EcoRI fragment in the wild-

type allele, and a 7.8 kb EcoRI fragment in the mutated

allele.

Targeted embryonic stem cell clone E-53 was injected

into C57 ⁄B6 blastocysts to generate chimeric mice. Chime-

ric males were mated with C57 ⁄B6 females and progenies

were analysed for germline transmission of the mutated

allele. Unless otherwise specified, the experiments were per-

formed with 129 ⁄Sv-C57 ⁄B6 hybrid descendants (F1) of

these animals. Backcross into C57 ⁄B6 was also carried out

for four generations to obtain mice with > 90% C57 ⁄B6genetic background. All experiments involving animals were

conducted according to protocols approved by the

Bioethics Committee of University of Perugia.

Mice were genotyped for the introduced Tm7sf2 mutated

gene by PCR analysis of tail genomic DNA using three spe-

cific primers (a, 5¢-AAGGCTTTGGTAGCTCCTGCCT-3¢;b, 5¢-TGAGGCCAGGTCTCAGCTCAC-3¢; neo, 5¢-GCT

ATCAGGACATAGCGTTGGC-3¢; see Fig. 2B). PCR

cycling conditions were: 2 min denaturation at 95 �C fol-

lowed by 35 cycles of 30 s at 95 �C, 45 s at 65 �C, 30 s at

72 �C, and a final extension of 5 min at 72 �C. Mouse

genotype was confirmed by Southern blotting analysis.

5¢ RACE

The transcription start site of the mouse Tm7sf2 gene was

determined by 5¢-RACE [34]. Mouse liver RNA was reverse

transcribed using the primer 5¢-AGGAGCTACCAAAGC

CTTCG-3¢ (nucleotides +468 to +449 from the ATG start

codon). A homopolymeric A-tail was then added to the

3¢-end using terminal transferase and dATP. The tailing

product was purified and amplified using the nested reverse

primer 5¢-CGACTCTTGTCCTTCAGTTCC-3¢ (nucleotides+284 to +264) and an oligo(dT) forward primer. The

product of amplification was amplified again using the

nested reverse primer 5¢-GTGCAGGCAGCAAATAGA

GC-3¢ (nucleotides +244 to +225) and an oligo(dT)

forward primer. The PCR product was analysed on 1.2%

agarose gel and sequenced in both strands.

Preparation of subcellular fractions from mouse

liver

Subcellular fractions were obtained from liver of 6-week-old

mice. Microsomes were prepared as previously described

[35,36] in the presence of complete protease inhibitor cock-

tail (Roche) and 0.03 mm phenylmethylsulfonyl fluoride.

Nuclei were isolated by the method of Kaufmann et al.

[37] and resuspended in 50 mm Tris ⁄HCl (pH 7.4), 0.25 m

sucrose, 5 mm MgSO4, containing 50% glycerol to avoid

freezing. For western blot analysis, the suspensions were

sonicated and the nuclear membrane fractions were recov-

ered by centrifuging at 100 000 g.

Preparations of microsomes and nuclei were not

cross-contaminated, as verified by western blotting with the

anti-(bovine C14SR) and anti-(human LBR) sera. Protein

concentration was determined by the method of Lowry [38].

Western blotting

Liver microsomal or nuclear membrane proteins were anal-

ysed by western blotting using polyclonal rabbit anti-

(bovine C14SR) serum or guinea-pig anti-(human LBR)

serum as previously described [5]. Equivalent protein load-

ing was checked by Ponceau staining of poly(vinylidene



difluoride) membranes after protein transfer. Labelled pro-

teins were detected by the enhanced chemiluminescence

assay, images were acquired using the VersaDoc Imaging

System, and signals were quantified using quantity one

software (Bio-Rad, Milan, Italy).

3b-Hydroxysterol D14-reductase activity

Determination of 3b-hydroxysterol D14-reductase activity

was performed by incubating the enzyme source for 30 min

at 37 �C with 5a-cholesta-8,14-dien-3b-ol (C27D8,14) [39]. To

measure the C27D8,14 substrate and the 5a-cholesta-8-en-3b-ol product (C27D8), sterols were purified and analysed by

GC-MS using 5a-cholestane internal standard as previously

described [2]. 3b-Hydroxysterol D14-reductase activity was

evaluated on the basis of peak area ratios between m ⁄ z 426

and m ⁄ z 372 ions (C27D8,14 ⁄ 5a-cholestane) or m ⁄ z 428 and

m ⁄ z 372 ions (C27D8 ⁄ 5a-cholestane) at the expected reten-

tion time. GC-MS analysis of sterols did not reveal reaction

products other than C27D8. In all conditions, C27D8 sterol

was undetectable at zero incubation time.

Sterol determinations

Sterols in liver microsomal membrane preparations were

evaluated by GC-MS. Lipids were saponified with methan-

olic KOH and sterols were extracted and analysed as

described previously [2]. Cholesterol was determined as

peak area ratio between m ⁄ z 368 and m ⁄ z 372 ions (choles-

terol ⁄ 5a-cholestane) using a calibration curve. Alterna-

tively, lipids were extracted by the method of Folch et al.

[40] and saponified. Cholesterol was separated by thin-layer

chromatography (n-hexane ⁄ diethyl ether ⁄ acetic acid,

70 : 30 : 1, v ⁄ v ⁄ v), visualized with Cu-acetate reagent [41],

and quantified as described for western blotting. Purified

cholesterol standard was run on the same plate as the sam-

ples to construct calibration curves.

Plasma cholesterol was determined enzymatically with a

commercial kit (Cholesterol Liquid, Sentinel, Milan, Italy).

RNA extraction and qRT-PCR analysis

Pooled tissues from three 8-week-old male mice (females

were used for ovary) in RNAlater RNA Stabilization

Reagent (Qiagen) were homogenized using Qiazol Lysis

Reagent (Qiagen). Total RNA was extracted according to

manufacture’s instructions and then subjected to clean up

on mini columns (RNeasy Mini Kit, Qiagen). RNA was

reverse transcribed using AffinityScript Multiple Tempera-

ture Reverse Transcriptase and random hexamer primers.

qRT-PCR amplifications were performed using Mx3000P�Real-Time PCR System with Brilliant� SYBR� Green

QPCR Master Mix (Stratagene) and ROX as reference dye.

Tm7sf2 specific primers were: forward, 5¢-GCCTCGGTTC

CTTTGACTTC-3¢; reverse, 5¢-CCATTGACCAGCCACAT

AGC-3¢. Lbr specific primers were: forward, 5¢-GTGCTCC

TGAGTGCTTAC-3¢; reverse, 5¢-GCCAATGAAGAAGT

CGTAC-3¢. Housekeeping control was mouse Hprt [42].

Experiments were performed in triplicate and repeated

twice with different RNA preparations. Results were

analysed using mx3000p�system software (Stratagene).

The specificity of the amplified products was assessed by

melting curve.

Affymetrix oligonucleotide array hybridization

and data analysis

Gene expression analysis was performed in liver of

6-week-old wild-type and knockout male mice after the

fourth backcross generation. Total RNA of pooled liver

tissue from three animals was extracted as described

above.

Probe synthesis, hybridization, and data analysis were

performed at the Affymetrix Microarray Unit (Campus

IFOM IEO, Milan, Italy). Biotin-labelled cRNA targets

were synthesized starting from 3 lg of total RNA. Double

stranded cDNA synthesis was performed with One-Cycle

cDNA Synthesis Kit, and biotin-labelled antisense RNA

was transcribed in vitro using IVT Labelling Kit. All steps

of the labelling protocol were performed with One-Cycle

Eukaryotic Target Labelling Assay as suggested by Affyme-

trix (http://www.affymetrix.com/support/technical/manual/

expression_manual.affx). Hybridization parameters were set

according to Affymetrix. Two copies of the complete gene-

chipmoe430 2.0 were hybridized with each biotin-labelled

target. Images were scanned using an Affymetrix GeneChip

Scanner3000 7G, using default parameters. The resulting

images were analysed using genechip operating software

v1.4 (GCOS1.4).

Some differentially expressed genes were analysed by

qRT-PCR as described above using the following primers.

Dbp: forward, 5¢-CTCGCCCTGTCAAGCATTCC-3¢;reverse, 5¢-TGATTGGTTGAGGCTTCAGTTCC-3¢. Sdc4:

forward, 5¢-GCGGCTCGGATGACTTTG-3¢; reverse, 5¢-AAGGGCTCAATCACTTCAGG-3¢. Cib3: forward, 5¢-ATGACTTCAACAATGACAACTAC-3¢; reverse, 5¢-ATC

CAGCACCTTCTCACAG-3¢. Cyp4a10 ⁄ 31: forward, 5¢-GC

CTCTGTGCTCGGTCTG-3¢; reverse 5¢-AGCCTTGAGTA

GCCATTGCC-3¢. Cyp2c39: forward 5¢-TGCTCTCCTAC

TCCTGATGAAG-3¢; reverse, 5¢-GGGCATGTGGTTCCT

GTCC-3¢. Cdc20: forward, 5¢-GCAACAGGAGGAGGA

ACCAG-3¢; reverse, 5¢-CATCCACAGCACTCAGACAG

G-3¢. Cdkn1a: forward, 5¢-AAAGTGTGCCGTTGTCTC-3¢;reverse, 5¢-AAAGTTCCACCGTTCTCG-3¢. Accn5: for-

ward, 5¢-GGTGACCATCCGCCAACTG-3¢; reverse, 5¢-CCGTAAGTGCTGTAGGTAATGAAG-3¢. Onecut1: forward,

5¢-CCTCTATGAATAACCTCTATACC-3¢; reverse, 5¢-TGCTGGGAGTTGTGAATG-3¢.



Acknowledgements

The financial support of Telethon–Italy (Grant no.

GGP030102) is gratefully acknowledged. We would

like to thank Aurelio Toia for assistance in GC-MS

analysis of sterols.

References

1 Holmer L, Pezhman A & Worman HJ (1998) The

human lamin B receptor ⁄ sterol reductase multigene

family. Genomics 54, 469–476.

2 Roberti R, Bennati AM, Galli G, Caruso D, Maras B,

Aisa C, Beccari T, Della Fazia MA & Servillo G (2002)

Cloning and expression of sterol delta14-reductase from

bovine liver. Eur J Biochem 269, 283–290.

3 Silve S, Dupuy P-H, Ferrara P & Loison G (1998)

Human lamin B receptor exhibits sterol C14-reductase

activity in Saccharomyces cerevisiae. Biochim Biophys

Acta 1392, 233–244.

4 Prakash A, Sengupta S, Aparna K & Kasbekar DP

(1999) The erg-3 (sterol D14,15-reductase) gene of

Neurospora crassa: generation of null mutants by

repeat-induced point mutation and complementation by

proteins chimeric for human lamin B receptor

sequences. Microbiology 145, 1443–1451.

5 Bennati AM, Castelli M, Della Fazia MA, Beccari T,

Caruso D, Servillo G & Roberti R (2006) Sterol depen-

dent regulation of human TM7SF2 gene expression:

role of the encoded 3b-hydroxysterol D14-reductase in

human cholesterol biosynthesis. Biochim Biophys Acta

1761, 677–685.

6 Wassif CA, Brownson KE, Sterner AL, Forlino A, Zer-

fas PM, Wilson WK, Starost MF & Porter FD (2007)

HEM dysplasia and ichthyosis are likely laminopathies

and not due to 3b-hydroxysterol D14-reductase defi-

ciency. Hum Mol Genet 16, 1176–1187.

7 Horton JD, Shah NA, Warrington JA, Anderson NN,

Park SW, Brown MS & Goldstein JL (2003) Com-

bined analysis of oligonucleotide microarray data from

transgenic and knockout mice identifies direct SREBP

target genes. Proc Natl Acad Sci USA 100, 12027–

12032.

8 Maxwell KN, Soccio RE, Duncan EM, Sehayek E &

Breslow JL (2003) Novel putative SREBP and LXR tar-

get genes identified by microarray analysis in liver of

cholesterol-fed mice. J Lipid Res 44, 2109–2119.

9 Greenberg CR, Rimoin DL, Gruber HE, DeSa DJ,

Reed M & Lachman RS (1988) A new autosomal reces-

sive lethal chondrodystrophy with congenital hydrops.

Am J Med Genet 29, 623–632.

10 Kelley RI & Herman GE (2001) Inborn errors of sterol

biosynthesis. Annu Rev Genomics Hum Genet 2, 299–

341.

11 Waterham HR, Koster J, Mooyer P, van Noort G, Kel-

ley RI, Wilcox WR, Wanders RJA, Hennekam RCM &

Oosterwijk JC (2003) Autosomal recessive HEM ⁄Greenberg skeletal dysplasia is caused by 3b-hydroxys-terol D14-reductase deficiency due to mutations in the

lamin B receptor gene. Am J Hum Genet 72, 1013–1017.

12 Schultz LD, Lyons BL, Burzenski LM, Gott B, Samuels

R, Schweitzer PA, Dreger C, Herrmann H, Kalscheuer

V, Olins AL et al. (2003) Mutations at the mouse ich-

thyosis locus are within the lamin B receptor gene: a

single gene model for human Pelger-Huet anomaly.

Hum Mol Genet 12, 61–69.

13 Irizarry RA, Bolstad BM, Collin F, Cope LM, Hobbs

B & Speed TP (2003) Summaries of Affymetrix Gene-

Chip probe level data. Nucleic Acids Res 31, e15.

14 Porter FD (2002) Malformation syndromes due to

inborn errors of cholesterol synthesis. J Clin Invest 110,

715–724.

15 Porter JA, Young KE & Beachy PA (1996) Cholesterol

modification of hedgehog signaling proteins in animal

development. Science 274, 255–259.

16 Tozawa R, Ishibashi S, Osuga J, Yagyu H, Oka T,

Chen Z, Ohashi K, Perrey S, Shionoiri F, Yahagi N

et al. (1999) Embryonic lethality and defective neural

tube closure in mice lacking squalene synthase. J Biol

Chem 274, 30843–30848.

17 Liu XY, Dangel AW, Kelley RI, Zhao W, Denny P,

Botcherby M, Cattanach B, Peters J, Hunsicker PR,

Mallon AM et al. (1999) The gene mutated in bare

patches and striated mice encodes a novel 3-beta-

hydroxysteroid dehydrogenase. Nat Genet 22, 182–

187.

18 Derry JM, Gormally E, Means GD, Zhao W, Meindl

A, Kelley RI, Boyd Y & Herman GE (1999) Mutations

in a D8-D7 sterol isomerase in the tattered mouse and

X-linked chondrodysplasys punctata. Nat Genet 22,

286–290.

19 Wassif CA, Zhu P, Kratz L, Krakowiak PA, Battaile

KP, Weight FF, Grinberg A, Steiner RD, Nwokoro

NA, Kelley RI et al. (2001) Biochemical, phenotypic

and neurophysiological characterization of a genetic

mouse model of RSH ⁄ Smith–Lemli–Opitz syndrome.

Hum Mol Genet 10, 555–564.

20 Fitzky BU, Moebius FF, Asaoka H, Waage-Baudet H,

Xu L, Xu G, Maeda N, Kluckman K, Hiller S, Yu H

et al. (2001) 7-Dehydrocholesterol-dependent proteolysis

of HMG-CoA reductase suppresses sterol biosynthesis

in a mouse model of Smith-Lemli-Opitz ⁄RSH syn-

drome. J Clin Invest 108, 905–915.

21 Ohashi K, Osuga J, Tozawa R, Kitamine T, Yagyu H,

Sekiya M, Tomita S, Okazaki H, Tamura Y, Yahagi N

et al. (2003) Early embryonic lethality caused by tar-

geted disruption of the HMG-CoA reductase gene.

J Biol Chem 278, 42936–42941.



22 Krakowiak PA, Wassif CA, Kratz L, Cozma D, Kavar-

ova M, Harris G, Grinberg A, Yang Y, Hunter AGW,

Tsokos M et al. (2003) Lathosterolosis, an inborn error

of human and murine cholesterol synthesis due to

lathosterol 5-desaturase deficiency. Hum Mol Genet

12, 1631–1641.

23 Mirza R, Hayasaka S, Takagishi Y, Cambe F, Ohmori

S, Maki K, Yamamoto M, Murakami K, Kaji T, Zad-

worny D et al. (2006) DHCR24 gene knockout mice

demonstrate lethal dermopathy with differentiation and

maturation defects in the epidermis. J Invest Dermatol

126, 638–647.

24 Holmer L & Worman HJ (2001) Inner nuclear mem-

brane proteins: functions and targeting. Cell Mol Life

Sci 58, 1741–1747.

25 Ellenberg J, Siggia ED, Moreira JE, Smith CL, Presley

JF, Worman HJ & Lippincot-Schwartz J (1997) Nuclear

membrane dynamics and reassembly in living cells: tar-

geting of an inner nuclear membrane protein in inter-

phase and mitosis. J Cell Biol 138, 1193–1206.

26 Hoffmann K, Sperling K, Olins AL & Olins DE (2007)

The granulocyte nucleus and lamin B receptor: avoiding

the ovoid. Chromosoma 116, 227–235.

27 Zwerger M, Herrmann H, Gaines P, Olins AL & Olins

DE (2008) Granulocytic nuclear differentiation of lam-

in B receptor-deficient mouse EPRO cells. Exp Hematol

36, 977–987.

28 Martınez-Botas J, Ferruelo AJ, Suarez Y, Fernandez C,

Gomez-Coronado D & Lasuncion MA (2001) Dose-

dependent effects of lovastatin on cell-cycle progression.

Distinct requirement of cholesterol and non-sterol meval-

onate derivatives. Biochim Biophys Acta 1532, 185–194.

29 Fernandez C, Lobo M, Gomez-Coronado D & Las-

uncion MA (2004) Cholesterol is essential for mitosis

progression and its deficiency induces polyploid cell for-

mation. Exp Cell Res 300, 109–120.

30 Fernandez C, Martın M, Gomez-Coronado D & Las-

uncion MA (2005) Effects of distal cholesterol biosyn-

thesis inhibitors on cell proliferation and cell-cycle

progression. J Lipid Res 46, 920–929.

31 Wagner N, Weber D, Seitz S & Krohne G (2004) The

lamin B receptor of Drosophila melanogaster. J Cell Sci

117, 2015–2028.

32 Dreger CK, Konig AR, Spring H, Lichter P & Herr-

mann H (2002) Investigation of nuclear architecture

with a domain-presenting expression system. J Struct

Biol 140, 100–115.

33 Magin TM, McWir J & Melton DW (1992) A new

mouse embryonic stem cell line with good germ line

contribution and gene targeting frequency. Nucleic

Acids Res 20, 3795–3796.

34 Frohman MA (1993) Rapid amplification of comple-

mentary DNA ends for generation of full-length com-

plementary DNAs: thermal RACE. Methods Enzymol

218, 340–356.

35 Mancini A, Del Rosso F, Roberti R, Orvietani P,

Coletti L & Binaglia L (1999) Purification of ethanol-

aminephosphotransferase from bovine liver microsomes.

Biochim Biophys Acta 1437, 80–92.

36 Roberti R, Mancini A, Freysz L & Binaglia L (1992)

Reversibility of the reactions catalyzed by cholinephos-

photransferase and ethanolaminephosphotransferase

solubilized from rat brain microsomes. Biochim Biophys

Acta 1165, 183–188.

37 Kaufmann SH, Gibson W & Shaper JH (1983) Charac-

terization of the major polypeptides of the rat liver

nuclear envelope. J Biol Chem 258, 2710–2719.

38 Lowry OH, Rosebrough NJ, Farr AL & Randall RJ

(1951) Protein measurement with the Folin phenol

reagent. J Biol Chem 193, 265–275.

39 Paik Y-K, Trzaskos JM, Shafiee A & Gaylor JL (1984)

Microsomal enzymes of cholesterol biosynthesis from

lanosterol. Characterization, solubilization, and partial

purification of NADPH-dependent D8,14-steroid14-reductase. J Biol Chem 259, 13413–13423.

40 Folch J, Lees M & Sloane Stanley GH (1957) A

simplified method for the isolation and purification of

total lipids from animal tissues. J Biol Chem 226, 497–

509.

41 Macala LJ, Yu RK & Ando S (1983) Analysis of brain

lipids by high performance thin-layer chromatography

and densitometry. J Lipid Res 24, 1243–1250.

42 Pieroni S, Della Fazia MA, Castelli M, Piobbico D,

Bartoli D, Brunacci C, Bellet MM, Viola-Magni M &

Servillo G (2008) HOPS is an essential constituent of

centrosome assembly. Cell Cycle 7, 1462–1466.



Disruption of the gene encoding 3β-hydroxysterol Δ14-reductase (Tm7sf2) in mice does not impair cholesterol biosynthesis

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