Báo cáo khoa học: SLC39A14, a LZT protein, is induced in adipogenesis and transports zinc pptx

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

a master regulator. One of the C ⁄ EBPs, C ⁄ EBPa,is
a target of PPARc.C⁄ EBPa positively activates
PPARc expression to maintain the differentiated state
[7]. The expression of C ⁄ EBPb and C ⁄ EBPd is
observed in the earliest period in differentiation. The
major function of C ⁄ EBPb and C ⁄ EBPd is the induc-
tion of expression of C ⁄ EBPa and PPARc [8]. Sterol
regulatory element-binding protein 1 (SREBP-1) is a
factor which binds to sterol regulatory elements of
cholesterol regulatory genes, regulating adipogenesis
through the production of ligand for PPARc [9].
Accordingly, C ⁄ EBPb and C ⁄ EBPd are thought to be
the factors initiating adipocyte differentiation. How-
ever, the expression of these factors is observed from
the mid to late phase of the differentiation, and the
earliest step in the differentiation into adipocytes
remains unknown.
Keywords
3T3-L1 cells; adipocyte differentiation; LIV
subfamily of ZIP transporters; SLC39A14;
Zrt ⁄ Irt-like protein
Correspondence
M. Imagawa, Department of Molecular
Biology, Graduate School of Pharmaceutical
Sciences, Nagoya City University, 3–1
Tanabe-dori, Mizuho-ku, Nagoya,
Aichi 467–8603, Japan
Tel ⁄ Fax: +81 52 836 3455
E-mail:
(Received 9 October 2004, revised 14

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

(TCL ⁄ TC10bL), p68 RNA helicase, Bach1 and
ARA70 is positively regulated in the initiation of adipo-
genesis [10–14]. Moreover, RGS2, TCL ⁄ TC10bL, and
p68 RNA helicase were identified as accelerating fac-
tors of adipocyte differentiation [12–14]. Of the 102
genes, 46 seem to be unknown whose functions
remain unclear. Therefore, we have focused on these
unidentified genes.
In this study, we report the cloning and characteri-
zation of one gene which we named fad123 (factor for
adipocyte differentiation-123). However, several recent
studies show that fad123 is identical to SLC39A14,a
member of the LZT (LIV-1 subfamily of ZIP zinc
transporters) subfamily of ZIP (Zrt ⁄ Irt-like proteins)
transporters [15,16]. SLC39A14 expression was eleva-
ted during the adipogenesis of mouse 3T3-L1 cells.
Moreover, expression was highly restricted to the dif-
ferentiation state of 3T3-L1 cells, because high level
expression was observed in growth-arrested 3T3-L1
cells, and the expression level was quite low in prolifer-
ating 3T3-L1 cells or nonadipogenic NIH-3T3 cells,
which cannot differentiate into adipocytes.
SLC39A14 is a member of the LZT subfamily of
ZIP transporters, and the ZIP superfamily is reported
to have roles in zinc uptake [17–19]. To test this abil-
ity, we have established K562 cells expressing
SLC39A14 and demonstrated that SLC39A14 func-
tions as a zinc transporter. Our findings indicate that
SLC39A14 participates in the uptake of zinc during
adipocyte differentiation.

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.

Finally, the combined sequences of the subtracted frag-
ment and the fragments obtained by RT-PCR and
RACE resulted in a 3660-bp full-length cDNA frag-
ment of SLC39A14 with an ORF of 489 amino acids.
Recently, SLC39A14 was reported to belong to the
LZT proteins, one of four subfamilies of ZIP transpor-
ter [15,16]. The deduced amino acid sequence of
SLC39A14 is known to have eight transmembrane
regions widely conserved in ZIP transporters including
the LZT proteins [15]. However, four of the five
transmembrane region prediction software packages
‘SOSUI’, ‘TMpred’, ‘PSORT II’, ‘HMMTOP’ and
‘DAS’, predicted a ninth transmembrane region at the
N-terminal end [(*) Fig. 1B]. In the loop between the
fourth and fifth transmembrane regions, there is a histi-
dine-rich repeat HHHGHSHY with the general formula
(HX)n, where n ¼ 3–6 [15]. Histidine-rich repeats are
considered to be potential metal-binding domains
[17,20,21]. Although another zinc transporting domain,
HEXPHE, has also been found, the first histidine in
the HEXPHE domain is not conserved in SLC39A14
(EEFPHE) as already reported [15] (Fig. 1B). HNF
motif which is highly conserved in LZT subfamily was
conserved in the fifth transmembrane region as it is also
already reported [15] (Fig. 1B).
The mouse genome database was made public by
the Mouse Genome Sequencing Consortium [22].
Using this database, we aimed to identify the genomic
distribution of mouse SLC39A14. A BLAST search of
the mouse genome database was performed with the

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-

sion pattern is basically the same as in 3T3-L1 cells.
These results indicate that SLC39A14 is specifi-
cally expressed in the early stages of adipocyte
differentiation.
Expression profile of SLC39A14 in the adipocyte
differentiable state and nondifferentiable state
We next determined whether or not the expression of
SLC39A14 was restricted to the adipocyte differenti-
ation state. Mouse 3T3-L1 cells differentiate into adi-
pocytes in the presence of inducers when the growth of
the cells has been arrested. On the other hand, prolifer-
ating 3T3-L1 cells do not differentiate into adipocytes
even with stimulation by inducers. Mouse NIH-3T3
cells in either state do not differentiate into adipocytes
when stimulated with inducers. These two cell lines
were stimulated with inducers while in a growth-arres-
ted or proliferating state. Total RNA was prepared
from the cells before and 3 h after the stimulation.
Although the expression of SLC39A14 was observed
in growth-arrested 3T3-L1 cells and NIH-3T3 cells, it
was dominant in the former (Fig. 3). These results
indicate that the expression of SLC39A14 is restricted
to the adipocyte differentiable state.
(SU)
2518
3147
58
1693
(RT-1)
(RT-2)

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

HNF
A
B
C
Fig. 1. Schematic representation of mouse
SLC39A14. (A) Cloning of mouse SLC39-
A14. The full-length cDNA for mouse
SLC39A14 was isolated by RT-PCR,
5¢-RACE and 3¢-RACE. SU, RT-1–3, R-5¢-and
R-3¢ are fragments obtained from the
original PCR-subtraction, RT-PCR, 5¢-RACE
and 3¢-RACE, respectively. The combined
schematic structure is presented as
SLC39A14 and the start and stop positions
are indicated. The predicted amino acid
sequence revealed a 489-amino acid protein
for mouse SLC39A14. (B) The schematic
structure of mouse SLC39A14. The nine
transmembrane domains (according to
Taylor et al. [15] and five transmembrane
prediction software packages), histidine-rich
motif [(HX)n, n ¼ 3–6], LZT protein
conserved motif (HEXPHE) and (HNF) are
shown. H, histidine; E, glutamic acid; P,
proline and X, any amino acid. *Transmem-
brane domain, not reported previously.
(C) The predicted exon ⁄ intron structure of
mouse SLC39A14 from the Mouse Genome
Database. The positions of exons are
indicated. The start and stop positions are

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

hr day
03162412 4268
3T3-L1
40000
20000
0
Relative mRNA expression
hr day
03162412 4268
3T3-F442A
9000
4500
0
Relative mRNA expression
A
B
C
Fig. 2. Time course of SLC39A14 mRNA expression in the early
stages of adipocyte differentiation. (A) Northern blot analysis of
SLC39A14 in 3T3-L1 cells. Total RNA prepared at various time
points after treatment with adipogenic inducers was prepared from
3T3-L1 cells. Isolated total RNA (25 lg) was loaded and subjected
to northern blot analysis of SLC39A14. The subtracted cDNA frag-
ment from the PCR-subtraction method was used as a probe.
b-Actin is shown as a control. (B) Q-PCR analysis of SLC39A14
expression in 3T3-L1 cells. The expression level of SLC39A14 was
determined at various time points in the differentiation of 3T3-L1
cells by Q-PCR and normalized with 18S rRNA expression deter-
mined by Q-PCR. Each column represents the mean with SD (n ¼
3). (C) Q-PCR analysis of SLC39A14 expression in 3T3-F442A cells.

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-

ted [17,18]. Moreover, K562 cells can grow in suspen-
sion culture, which simplifies the assay.
First, we determined the subcellular localization of
mouse SLC39A14 in K562 cells when exogenously
transfected. The vector expressing EGFP-fused
SLC39A14 was transiently transfected to K562 cells
and the signals were detected with confocal scanning
laser microscopy. As shown in Fig. 5A, GFP-
SLC39A14 was found in the plasma membrane region.
When the empty vector was transfected as a control,
GFP signal was detected in the whole region of the
cell. GFP-SLC39A14 was also detected in some organ-
elles. However, the details remain to be investigated.
Next, we performed the zinc uptake assay. The
SLC39A14 ORF was subcloned into pBK-CMV and
transfected into the K562 cells. By selecting with
G418, we isolated cells which stably express
SLC39A14. As a control, an empty pBK-CMV vector
was transfected, and the cells were selected and iso-
lated in the same manner.
The expression level of exogenous SLC39A14 was
analyzed by northern blotting (Fig. 5B). The expres-
sion of SLC39A14 was only observed in the
SLC39A14-expressing K562 cells, not the control cells.
Using these stable transformants and
65
ZnCl
2
, the abil-
ity of SLC39A14 to accumulate zinc was determined

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

PPARc-deficient cells the ability to differentiate [26].
PPARc has been implicated as a crucial regulator
for adipocyte differentiation. C ⁄ EBPb and C ⁄ EBPd
both have the ability to activate the expression of
PPARc and C ⁄ EBPa [8]. The expression of these fac-
tors was observed prior to that of PPARc and
C ⁄ EBPa. However, it is observed from the mid-phase
of adipocyte differentiation, and the events occurring
prior to the expression of these master regulators are
not well understood.
We have previously isolated genes expressed transi-
ently during the early stages of adipocyte differenti-
ation [10,11]. Of these, RGS2, TCL ⁄ TC10bL and p68
RNA helicase were induced to express during the initi-
ation of adipocyte differentiation [12–14]. Further-
more, the ectopic expression of RGS2 or TCL ⁄
TC10bL accelerated the adipogenesis of a nonadipo-
genic cell line, NIH-3T3 [13,14]. These findings indica-
ted the existence of unknown molecular mechanisms
Brain
Heart
Adipocyte
Stromal-vascular
Testis
Kidney
Liver
Skeletal muscle
Lung
BAT
200000

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

150
100
50
0
65
Zn Accumulation
pmol/10
6
cells
[Zn] (µM)
5
4
3
2
1
0
65
Zn Uptake Rate
pmol/min/10
6
cells
6
**
***
*
**
***
SLC39A14
Control
SLC39A14

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

it to be SLC39A14. The deduced amino acid sequence
of mouse SLC39A14 consisted of 489 amino acids. As
a few extra bands were observed in the northern blot
of SLC39A14 in mouse RNA, there is a possibility of
the existence of isoforms of SLC39A14.
The human ortholog of SLC39A14 was reported as
BC015770 by Taylor et al. [15]. However, BC015770
has very weak similarity in its C-terminal end with the
mouse counterpart. XM046677 (also listed as D31887)
was also reported as a human ortholog of SLC39A14
by Eide [16]. XM046677 has high similarity as a whole,
and the methionine, which we confirmed as the first
methionine of mouse, was the 66th amino acid in
XM046677 (39th in D31887). The result of 5¢-RACE
indicated that cDNA no longer existed in front of the
first methionine of mouse SLC39A14. Additionally, in
the amino acid sequences around this methionine,
Kozak’s sequence was well conserved [27]. However,
during the preparation of this manuscript, XM046677
was withdrawn by NCBI. On the other hand, the locu-
slink site in NCBI suggests that human counterpart
for mouse SLC39A14 is NP_056174 or BAD18780.
However, these two sequences have less similarity
(44.4%) through 160 aa to 188 aa with mouse
SLC39A14, whereas XM046677 or D31887 has higher
similarity (96.5%) in same region. The human genome
database search revealed that this difference was result
of different usage of exon 4. Therefore, we still do not
have the exact sequence of the human ortholog of
SLC39A14, and the cloning of full-length cDNA for

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,

we have established a stable SLC39A14-expressing
transformant, and demonstrated that these cells signifi-
cantly accumulate zinc compared control cells.
During the earliest stages of the adipocyte differenti-
ation of 3T3-L1 cells, it is reported that zinc is accu-
mulated transiently. Moreover, when the accumulation
was blocked by the addition of a zinc chelator, mitotic
clonal expansion was inhibited [28]. Another interest-
ing feature of zinc is that it mimics the effect of insulin
on glucose transport, lipogenesis and leptin production
[29–31]. Recently, LIV1, one of the LZT proteins, was
identified as a downstream target of STAT3 which is
activated during the epithelial-mesenchymal transition
and has an essential role in cell proliferation and dif-
ferentiation in zebrafish [32]. Taken together, it is
strongly suggested that SLC39A14 plays an important
role in the uptake of zinc during the differentiation of
3T3-L1 cells into adipocytes. However, the molecular
mechanism behind the actions of SLC39A14 during
the adipogenesis of 3T3-L1 cells is still not clear.
Therefore, further studies using SLC39A14 knockout
cells are required.
Experimental procedures
Cloning of full-length cDNA of mouse SLC39A14
As mouse SLC39A14 cDNA was isolated as a small 640-bp
fragment, RT-PCR, 5¢-RACE and 3¢-RACE were used for
cloning the full-length cDNA. RT-PCR was performed with
ReverTra Ace (Toyobo Co., Ltd. Osaka, Japan) according to
the manufacturer’s directions. Total RNA was isolated from
3T3-L1 cells (Dainippon Pharmaceutical Co., Ltd. Osaka,

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

GAGGGT-3¢. The fragments obtained from RACE and
RT-PCR were subcloned into a T-added EcoRV site of
pBluescript KS +0.
DNA sequencing and database analysis
The sequence was determined with the automated sequencer
DSQ-1000 (Shimadzu Corp., Kyoto, Japan) and an ABI
PRISM 310 (Applied Biosystems, Foster City, CA, USA).
The database search for the prediction of mouse SLC39A14
was performed using a genome browser on the UCSC Gen-
ome Bioinformatics homepage ( />RNA isolation and northern blot analysis
Total RNA was extracted with TRIzol (Invitrogen, Carls-
bad, CA, USA) according to the manufacturer’s instruc-
tions. For northern blot analyses, 15–25 lg of total RNA
was electrophoresed on a 1% agarose gel containing 2%
formaldehyde, and then transferred to a Hybond-N+
nylon membrane (Amersham Pharmacia Biotech Ltd, Pis-
cataway, NJ, USA). Each probe was labeled with
[
32
P]dCTP[aP] using a BcaBEST labeling kit (Takara Bio-
medicals, Kusatsu, Japan).
Cell culture
Mouse 3T3-L1 (ATCC CL173) preadipocyte cells (Dainip-
pon Pharmaceutical Co., Ltd.) were maintained in Dul-
becco’s modified Eagle’s medium (DMEM) containing 10%
calf serum. For the differentiation experiment, the medium
was replaced with DMEM containing 10% fetal bovine
serum (FBS), 10 lgÆmL
)1
of insulin, 0.5 mm 3-isobutyl-

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

amounts of the unknown samples.
Fractionation of fat cells
The fat cells were prepared as described previously [33]. In
brief, epidermal fat pads were isolated from male C57Bl ⁄ 6J
mice (Japan SLC, Inc. Hamamatsu, Japan) aged 6 weeks,
killed by exposure to high concentrations of CO
2
, washed
with sterile NaCl ⁄ P
i
, minced, and washed with Krebs-Ringer
bicarbonate (KRB) buffer (pH 7.4). Then, the minced tissue
was digested with 1.5 mgÆmL
)1
of collagenase type II (Sigma-
Aldrich, Inc., St Louis, MO, USA) in KRB buffer, contain-
ing 4% bovine serum albumin at 37 °C for 1 h on a shaking
platform. The undigested tissue was removed with a 250 lm
nylon mesh and the digested fraction was centrifuged at
500 g for 5 min. The adipocytes were obtained from the
upper most layer, washed with buffer, and centrifuged to
remove other cells. The stromal-vascular cells were resus-
pended in erythrocyte lysis buffer [150 mm NH
4
Cl, 25 mm
NH
4
HCO
3
and 1 mm EDTA (pH 7.7)], filtered through

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.

The full-length cDNA of SLC39A14 was subcloned into
the vector pBK-CMV. pBK-CMV-SLC39A14 or pBK-
CMV empty vector was transfected to K562 cells by elec-
troporation. The stable transformants were selected in the
presence of 0.8 mgÆmL
)1
G418 containing Ham’s F12 (Invi-
trogen) supplied with 10% FBS for one week. Cells derived
from single clone were isolated, stored individually and
used for the
65
Zn uptake assay.
65
Zn uptake assay
65
ZnCl
2
(246 CiÆg
)1
, 1432.7 lCiÆmL
)1
of 0.5 m HCl) was
obtained from Isotope Products Laboratories (Valencia,
CA, USA). The
65
Zn uptake assay was conducted as
reported previously [17,18]. A ZnCl
2
stock solution was
prepared at 100 mm in 0.02 m HCl as described [17,18].

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

volume of cold stop buffer (15 mm Hepes, 100 mm glu-
cose, 150 mm KCl and 1 mm EDTA, pH 7.0). The cells
were centrifuged and washed with cold stop-buffer three
times. Then radioactivity was measured with a c-counter
ARC-7001 (ALOKA, Tokyo, Japan).
Acknowledgements
This study was supported in part by grants from the
Ministry of Education, Culture, Sports, Science and
Technology (MEXT), Japan, Japan Society for the
Promotion of Science (JSPS), and ONO Medical
Research Foundation, Japan.
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