SLC39A14, a LZT protein, is induced in adipogenesis
and transports zinc
Kei Tominaga
1,2
, Takeshi Kagata
1
, Yoshikazu Johmura
1
, Tomoaki Hishida
1
, Makoto Nishizuka
1
and Masayoshi Imagawa
1
1 Department of Molecular Biology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Aichi, Japan
2 Research Division, Nissui Pharmaceutical Co. Ltd, Hokunanmoro, Yuki, Ibaraki, Japan
Obesity is a major health problem in industrialised
societies. It is related to the development of type 2 dia-
betes mellitus, hypertension and arteriosclerosis [1].
Obesity often results in these kinds of life style-related
diseases as the balance of biologically active substances
such as leptin, tumor necrosis factor-a, adiponectin,
adipsin, and plasminogen activator inhibitor-1 secreted
from adipose tissue is disrupted [2–6].
During the differentiation of preadipocytes to adi-
pocytes, three classes of transcription factor proteins
are known to function as master regulators. Per-
oxisome proliferator activated receptor c (PPARc)
transactivates adipocyte-specific genes like those for
aP2 and lipoprotein lipase. The CCAAT ⁄ enhancer-
binding protein (C ⁄ EBP) family is also recognized as

December 2004, accepted 24 January 2005)
doi:10.1111/j.1742-4658.2005.04580.x
During adipocyte differentiation, there is an underlying complex series of
gene expressions. We have previously isolated many genes whose expres-
sion levels are quickly elevated by the addition of inducers to mouse 3T3-
L1 preadipocyte cells. Here we report the isolation and characterization of
SLC39A14, a member of the LZT proteins, one of the subfamilies of ZIP
transporters. The expression of the SLC39A14 gene was strongly and rap-
idly induced at the early stages of differentiation. Moreover, it was highly
restricted to the potential differentiation state of 3T3-L1 cells and the
expression level was quite low in the nonadipogenic NIH-3T3 cells, indica-
ting a dominant expression in adipocyte differentiation. The zinc uptake
assay revealed that SLC39A14 functions as a zinc transporter. Taken
together, these results suggest that SLC39A14 plays a role as a zinc trans-
porter during the early stages of adipogenesis.
Abbreviations
fad, factor for adipocyte differentiation; C ⁄ EBP, CCAAT ⁄ enhancer-binding protein; Dex, dexamethasone; DMEM, Dulbecco’s modified
Eagle’s medium; FBS, fetal bovine serum; IBMX, 3-isobutyl-1-methylxantine; LZT, LIV subfamily of ZIP transporters; PPARc, peroxisome
proliferator-activated receptor c; SREBP, sterol regulatory element-binding protein; ZIP, Zrt ⁄ Irt-like protein.
1590 FEBS Journal 272 (2005) 1590–1599 ª 2005 FEBS
As reported previously, we have isolated many
genes expressed in the earliest stages of adipocyte dif-
ferentiation some of which positively regulate the
differentiation [10,11]. Adipocyte hyperplasia is
mimicked by the mouse fibroblastic cell line 3T3-L1.
Using this cell line, 102 genes were isolated as up-
regulated in the earliest stage of the differentiation by
the PCR-subtraction cloning method [10,11]. We have
already reported that the expression of regulator of
G protein signaling 2 (RGS2), TC10-like ⁄ TC10bLong

Results
Cloning of full-length mouse SLC39A14 cDNA
In previous studies, we isolated 102 clones the expres-
sion of which is increased at 3 h after induction by the
PCR-subtraction cloning method. These include 46
unknown genes that were not listed in the database
[10,11]. In the present study, we first attempted to iso-
late a full-length cDNA of SLC39A14 using RT-PCR
and RACE. The cDNA fragment isolated by the
PCR-subtraction method was only 630 bp long, as the
amplified fragments were digested with RsaI to prevent
bias in subcloning [10]. Isolation of the cDNA of
SLC39A14 was performed by predicting the mouse
SLC39A14 full-length ORF by a database search
at UCSC Genome Bioinformatics (http://genome.
ucsc.edu/). The search results revealed the existence of
10 exons on mouse chromosome 14 in front of the
exon including a 630 bp subtracted SLC39A14 cDNA
fragment. As these 11 exons exist near each other, we
expected the ORF of SLC39A14 to be included in
them.
To test this hypothesis, we performed RT-PCR
against cDNA prepared from 3T3-L1 cells 3 h after
induction using primers designed from the predicted
sequence, and observed 1636 bp (Fig. 1A, RT-1),
1282-bp (Fig. 1A, RT-2), and 924 bp (Fig. 1A, RT-3)
cDNA fragments. We next performed 5¢-RACE and
3¢-RACE for isolation of the 5¢-end and 3¢-end of
SLC39A14. As a result, 935 bp (Fig. 1A, R-5¢) and
996 bp (Fig. 1A, R-3¢) cDNA fragments were isolated.

mouse SLC39A14 full-length cDNA sequence. The
result indicated that mouse SLC39A14 located at
14D1 of chromosome 14 constituted 11 exons and 10
K. Tominaga et al. SLC39A14 is expressed during adipogenesis
FEBS Journal 272 (2005) 1590–1599 ª 2005 FEBS 1591
introns. In the sequences of the exon ⁄ intron junctions,
the GT ⁄ AG rule was conserved in all cases except for
the last exon coding the 3¢-UTR region (Fig. 1C).
Expression of SLC39A14 during early stages
of adipogenesis
The time course of the expression of SLC39A14 in
3T3-L1 cells was determined by northern blot analysis
as shown in Fig. 2A. SLC39A14 expression was
induced rapidly after the addition of inducers and
declined until 24 h after induction. This result indicates
that SLC39A14 is transiently expressed in the early
stages of adipocyte differentiation. The expression level
of SLC39A14 throughout adipogenesis including the
late stages was determined by Q-PCR for the quantita-
tive analysis of SLC39A14 . The same expression pat-
tern in the early stages was obtained from the Q-PCR
assay, and the level of expression in the late stages
was relatively low (Fig. 2B). We next determined the
expression profile of SLC39A14 in 3T3-F442 cells,
which is another preadipocyte cell line. These cells do
not need IBMX and Dex to differentiate into adipo-
cytes. The expression of SLC39A14 was determined
by Q-PCR. As shown in Fig. 2C, the expression of
SLC39A14 was transiently induced by the addition of
insulin to confluent 3T3-F442A cells, and the expres-

2147
3428
(RT-3)
1419
2342
stop
ATG
(R-5')
1
935
(R-3')
2665
3660
1
1728
SLC39A14
262
489 aa
3660
Exon 1 432
Mouse
SLC39A14
ATG stop
56 - 9 1110
Transmembrane domain
489 aa
Mouse SLC39A14
*
HHHGHSHY
EEFPHE

also indicated.
SLC39A14 is expressed during adipogenesis K. Tominaga et al.
1592 FEBS Journal 272 (2005) 1590–1599 ª 2005 FEBS
Tissue distribution of SLC39A14
We next determined the expression of SLC39A14 in
brain, heart, skeletal muscle, kidney, lung, liver, testis,
epidermal white adipose tissue (WAT) and interscapu-
lar brown adipose tissue (BAT) isolated from adult
male mice by Q-PCR. WAT samples were fractionated
into stromal-vascular cells and mature adipocytes. As
shown in Fig. 4, strong expression was observed in
liver, whereas moderate expression was observed in
brain, heart, skeletal muscle, kidney and WAT. The
expression was almost undetectable in lung, testis and
BAT. Interestingly, the level of expression was higher
in the stromal–vascular fraction than in mature adipo-
cytes, suggesting that SLC39A14 expressed predo-
minantly in the preadipocytes than in the mature
adipocytes.
Characterization of SLC39A14 as a
zinc transporter
SLC39A14 is one of the LZT proteins that compose a
subfamily of ZIP zinc transporter proteins. Therefore,
we next attempted to investigate whether SLC39A14
031624120.5 2
28S
18S
hr
SLC39A14
β-actin

The expression level of SLC39A14 was determined at various time
points in the differentiation of 3T3-F442A cells by Q-PCR and
normalized with 18S rRNA expression determined by Q-PCR. Each
column represents the mean with standard deviation (n ¼ 3).
relative intensity
1200000
0
0
0033330
3T3-L1 NIH-3T3
growth
growth
arrested
arrested
proliferating
proliferating
hr
Fig. 3. Expression profile of SLC39A14 in differentiating and nondif-
ferentiating cells. Total RNA (25 lg) isolated from proliferating and
postconfluent (growth-arrested) 3T3-L1 and NIH-3T3 cells, before
and 3 h after induction with the inducers which are listed in the
experimental procedures, was loaded in each column. The subtrac-
ted cDNA fragment from the PCR-subtraction method was used as
a probe. Relative intensities are also shown (0–1 200 000).
K. Tominaga et al. SLC39A14 is expressed during adipogenesis
FEBS Journal 272 (2005) 1590–1599 ª 2005 FEBS 1593
functions as a zinc transporter. To this end, we used
human K562 erythroleukemia cells, known to be suit-
able for assaying the uptake of zinc, as a high level of
expression and proper protein localization were expec-

in the uptake buffer indicated in the Experimental pro-
cedures according to the methods of Gaither et al.
[17,18]. K562 cells have endogenous zinc uptake activ-
ity under the conditions outlined in the Experimental
procedures. However, the uptake of the SLC39A14-
expressing K562 cells was 2–3 fold higher than that
of the control cells at each concentration of zinc
(Fig. 5C). We next determined the accumulation of
zinc by the SLC39A14-expressing K562 cells. As
shown in Fig. 5D, the levels of zinc were significantly
elevated compared to those in the control cells. More-
over, when the same experiment was conducted at
4 °C, no uptake of zinc by SLC39A14-expressing K562
cells or control cells was detectable, indicating that the
accumulation was transporter-mediated rather than
due to the cell surface binding. These results strongly
suggest that SLC39A14 functions as a zinc transporter.
Discussion
Adipocyte differentiation is one of the most studied
models of differentiation. It is already known that
several transcription factors function in a complex
cascade. A key regulatory role for PPARc during
adipogenesis was demonstrated by gain of function
experiments, which showed that ectopic expression and
activation of PPARc in fibroblasts or myoblasts pro-
moted adipogenesis [23]. It has also been shown that
PPARc is necessary for adipocyte differentiation
in vivo [24]. C ⁄ EBPa was also shown to be a regulator
for adipocyte differentiation in gain of function experi-
ments [25]. However, C ⁄ EBPa could not restore to

100000
0
Relative mRNA expression
WAT
Fig. 4. Tissue distribution of SLC39A14. The expression level of
SLC39A14 in various tissues isolated from C57Bl ⁄ 6 J mice was
determined by Q-PCR and normalized with 18S rRNA expression
determined by Q-PCR. Stromal vascular cells and adipocytes were
fractionated from isolated white adipose tissue. Each column repre-
sents the mean with SD (n ¼ 3). WAT, white adipose tissue; BAT,
brown adipose tissue.
SLC39A14 is expressed during adipogenesis K. Tominaga et al.
1594 FEBS Journal 272 (2005) 1590–1599 ª 2005 FEBS
30
25
20
15
10
5
0
6050403020100
65
Zn Uptake Rate
pmol/min/10
6
cells
Time (min)
3020100
250
200

Control
SLC39A14
Control
EGFP
TLI
28S
18S
A
C
D
B
Fig. 5. Functional expression of SLC39A14 in K562 cells. (A) Intracellular localization of SLC39A14 in K562 cells. K562 cells transiently trans-
fected with EGFP-SLC39A14 (SLC39A14) or empty vector (control) were fixed and then the signals were detected with confocal laser scan-
ning microscopy. TLI, transmitted light image. (B) The ectopic expression of SLC39A14 in a stable transformant of K562 cells. Northern blot
analysis was performed for RNAs prepared from pCMV-SLC39A14-expressing K562 cells and control cells transfected with empty vector.
The full-length cDNA of SLC39A14 was used as a probe for northern blot analysis of expression level of SLC39A14 in K562 cells. The exo-
genous expression is shown. (C) Zinc uptake was assayed using pCMV-SLC39A14-expressing K562 cells (m) and control cells transfected
with empty vector (d). The cells were added to uptake buffer containing
65
Zn. (D) Zinc accumulation was assayed in SLC39A14-expressing
cells and control cells with 10 l
M
65
Zn at 37 °C (filled symbols) and at 4 °C (unfilled symbols) (left panel). The zinc uptake rate 30 min after
the accumulation started is shown in the right panel. For all panels, bars and plots denote the mean with SD (n ¼ 3); *P < 0.05; **P < 0.01;
***P < 0.001 comparing SLC39A14-expressing cells with control cells.
K. Tominaga et al. SLC39A14 is expressed during adipogenesis
FEBS Journal 272 (2005) 1590–1599 ª 2005 FEBS 1595
underlying the initiation of adipogenesis. In this study,
we have isolated and characterized fad123, and found

human SLC39A14 remains to be investigated.
SLC39A14 is a member of the LZT proteins, one of
the subfamilies of ZIP transporters, and is transiently
expressed upon stimulation with inducers of adipogene-
sis. Its expression was restricted to the adipocyte differ-
entiable state. Therefore, we have performed RNAi
experiments to knock down the expression of
SLC39A14 in differentiating 3T3-L1 cells. Although the
expression of SLC39A14 was suppressed by RNAi, the
ability of 3T3-L1 cells to differentiate was not affected
(data not shown). However, as SLC39A14 is part of a
large family of ZIP transporters, it is possible that other
members may substitute for the function of SLC39A14.
Further study on the functions of SLA39A14 in adipo-
cyte differentiation is definitely needed.
Zinc is an essential metal in all eukaryotes. Zinc
transporting proteins were first reported in yeast and
plants. In mammals, the ZIP superfamily is the most
studied zinc transporter. Human zip1 and zip2 are
reported to function as a zinc transporter by Gaither
et al. [17,18]. Transient transfection of three mouse
zips(zip1, zip2 and zip3) was demonstrated by Beattie
et al. [19], and it was indicated that these factors also
function as zinc transporters. It was reported that
SLC39A14 has no zinc transporting activity as it lacks
the initial H of the HEXXH motif, which is crucial for
the transport [15]. However, an analysis of the primary
structure of SLC39A14 indicated that this gene has
another crucial motif, a histidine-rich repeat, which is
a potential metal binding motif [17,20,21]. Therefore,

Japan) 3 h after induction as described below. The single
stranded cDNA was synthesized using a random primer and
ReverTra Ace. The PCR was performed with KOD plus
(Toyobo Co., Ltd), a SLC39A14-specific forward primer:
5¢-CCCACTCAGTAGCTGTGT-3¢,5¢-CAATGCTGGCAT
GAGCAT-3¢ or 5¢-CTTCTTGGGGAAACATG-3¢, and a
reverse primer: 5¢-CCAGCATAATGGAGAAGC-3¢,5¢-AA
CTGGACCCTAAGCCTA-3¢ or 5¢-ACTGGATCCTAGGT
GATC-3¢.5¢-RACE was performed using a Marathon cDNA
Amplification Kit (BD Biosciences Clontech, Palo Alto,
CA, USA) following the instructions of the manufacturer.
SLC39A14 is expressed during adipogenesis K. Tominaga et al.
1596 FEBS Journal 272 (2005) 1590–1599 ª 2005 FEBS
Total RNA was prepared from 3T3-L1 cells 3 h after
induction. mRNA was isolated from total RNA using
Oligotex-dT30 (Daiichi Pure Chemicals, Tokyo, Japan)
according to the manufacturer’s directions. The single
stranded cDNA was amplified with oligo-(dT) primer and
AMV reverse transcriptase. The second strand of cDNA
was synthesized using a second-strand enzyme cocktail con-
taining RNase H, Escherichia coli DNA polymerase I, and
E. coli DNA ligase. The resultant double-stranded cDNA
was ligated to a Marathon cDNA adapter by T4 DNA
ligase. The PCR for 5¢-RACE was performed using the for-
ward primer AP-1: 5¢-CCATCCTAATACGACTCACTAT
AGGGC-3¢ and a SLC39A14-specific reverse primer:
5¢-AACACCACTGCAGACTTGGAGACG-3¢. The PCR
for 3¢-RACE was performed using the forward primer AP-1:
5¢-CCATCCTAATACGACTCACTATAGGGC-3¢ and a
SLC39A14-specific reverse primer: 5¢-GATTGTAGGTCT

1-methylxantine (IBMX) and 1 lm dexamethasone (Dex) at
2 days post-confluence. After 2 days, cells were transferred
to DMEM containing 5 lgÆmL
)1
of insulin and 10% FBS,
then the cells were refed every 2 days. Mouse 3T3-F442A
(ECACC 70654) cells were maintained in DMEM contain-
ing 10% calf serum. For the differentiation experiment, the
medium was replaced with DMEM containing 10% FBS
and 5 lgÆmL
)1
of insulin when the cells were confluent. The
cells were refed every 2 days. Mouse NIH-3T3 (clone 5611,
JCRB 0615) fibroblastic cells were maintained in DMEM
containing 10% calf serum. K562 (RIKEN Cell Bank,
RCB No. RCB0027) cells were maintained in Ham’s F12
(Invitrogen) containing 10% FBS.
Real-time quantitative RT-PCR (Q-PCR)
The isolation and reverse transcription of total RNA were
done as described above. The ABI PRISM 5700 sequence
detection system (Applied Biosystems) was used to perform
Q-PCR. The predesigned primers and probe sets for
SLC39A14 and 18S rRNA were obtained from Applied
Biosystems. The reaction mixture was prepared using a
TaqMan Universal PCR Master Mix (Applied Biosystems)
according to the manufacturer’s instructions. The mixture
was incubated at 50 ° C for 2 min and at 95 °C for 10 min,
and then the PCR was conducted at 95 °C for 15 s and at
60 °C for 1 minute for 40 cycles. Relative standard curves
were generated in each experiment to calculate the input

28 lm nylon mesh and then precipitated at 500 g for 5 min.
All of our animal experiments were done in compliance with
Guidelines for the Care and Use of Laboratory Animals of
Nagoya City University Medical School.
Subcellular localization of SLC39A14 fused
to enhanced green fluorescent protein (EGFP)
The pEGFP-SLC39A14 chimeric plasmid was constructed
by subcloning the coding region into the 3¢-end of pEGFP-
K. Tominaga et al. SLC39A14 is expressed during adipogenesis
FEBS Journal 272 (2005) 1590–1599 ª 2005 FEBS 1597
C1 (BD Biosciences Clontech, Palo Alto, CA, USA)
in-frame. Transfection of EGFP-fusion protein expression
vector into K562 cells was performed by Nucleofector
(Amaxa, Cologne, Germany) using Cell Line Nucleofector
Kit V (Amaxa). K562 cells were harvested and resuspended
in Nucleofector solution at 1.0 · 10
6
cells per 100 lL. After
addition of 5 lg of expression vector, the cells were trans-
fected by program ‘T-16’ of Nucleofector. Then, the cells
were spread to 12-well plate. The transfected K562 cells
were harvested, washed with NaCl ⁄ P
i
and fixed in cold
methanol, and EGFP signal was detected by confocal laser
scanning microscopy.
Establishment of SLC39A14-expressing stable
transformants
The K562 cells that stably express SLC39A14 were estab-
lished by limiting dilution method using G418 selection.

A dilution was made to obtain 6, 20, 60 and 120 lm zinc
solution in uptake buffer (15 mm Hepes, 100 mm glucose
and 150 mm KCl, pH 7.0). Then, the trace amount of
65
ZnCl
2
was added to this solution. For the equilibration
of the zinc solution containing
65
Zn with other compo-
nents of the medium, the mixture was incubated at 25 °C
for 24 h before the experiment. The cells were grown to
25% confluence, harvested by centrifugation at 150 g for
3 min at 4 °C, and washed in cold uptake buffer. The
cells were resuspended in the prewarmed uptake buffer
(5 · 10
4
Æ250 lL
)1
), and incubated for 10 min at 37 °C.
Then, the cells were mixed with the same volume of
uptake buffer containing
65
ZnCl
2
(the final concentration
of ZnCl
2
was 3, 10, 30 and 60 lm) and incubated. The
uptake reaction was stopped by the addition of an equal

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