SLC39A14, a LZT protein, is induced in adipogenesis and transports zinc

SLC39A14, a LZT protein, is induced in adipogenesisand transports zincKei Tominaga1,2, Takeshi Kagata1, Yoshikazu Johmura1, Tomoaki Hishida1, Makoto Nishizuka1

and Masayoshi Imagawa1

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: [email protected]

(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, andp68 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

stopATG

(R-5')1 935

(R-3')2665 3660

1 1728

SLC39A14

262

489 aa

3660

Exon 1 432

Mouse SLC39A14

ATG stop

5 6 - 9 1110

Transmembrane domain

489 aaMouse SLC39A14

*HHHGHSHY EEFPHEHNF

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.


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

0 31 6 24120.5 2

28S

18S

hr

SLC39A14

β-actin

hr day

0 31 6 2412 42 6 8

3T3-L140000

20000

0

Re

lati

ve

mR

NA

ex

pre

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ion

hr day

0 31 6 2412 42 6 8

3T3-F442A9000

4500

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B

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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 ¼3). (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).

rela

tive

inte

nsi

ty

1200000

0

0 00 3333 0

3T3-L1 NIH-3T3

grow

th

grow

th

arre

sted

arre

sted

prol

ifera

ting

prol

ifera

ting

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).



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 65ZnCl2, 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 ⁄EBPdboth 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

Bra

in

Hea

rt

Ad

ipo

cyte

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lar

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is

Kid

ney

Liv

er

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l mu

scle

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ng

BA

T

200000

100000

0Rel

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RN

A e

xpre

ssio

n

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.



30

25

20

15

10

5

0

6050403020100

65Z

n U

pta

ke R

ate

pm

ol/m

in/1

06 c

ells

Time (min)

3020100

250

200

150

100

50

0

65Z

n A

ccu

mu

lati

on

pm

ol/1

06 c

ells

[Zn] (µM)

5

4

3

2

1

0

65Z

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ate

pm

ol/m

in/1

06 c

ells

6 **

***

*

**

***

SLC

39A

14C

ontro

l

SLC

39A

14C

ontro

l

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 65Zn. (D) Zinc accumulation was assayed in SLC39A14-expressing

cells and control cells with 10 lM65Zn 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.



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.



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 (http://genome.ucsc.edu/).

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

[32P]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 ⁄ 6 J

mice (Japan SLC, Inc. Hamamatsu, Japan) aged 6 weeks,

killed by exposure to high concentrations of CO2, washed

with sterile NaCl ⁄Pi, 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 lmnylon 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 NH4Cl, 25 mm

NH4HCO3 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-



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 · 106 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 ⁄Pi 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 65Zn uptake assay.

65Zn uptake assay

65ZnCl2 (246 CiÆg)1, 1432.7 lCiÆmL)1 of 0.5 m HCl) was

obtained from Isotope Products Laboratories (Valencia,

CA, USA). The 65Zn uptake assay was conducted as

reported previously [17,18]. A ZnCl2 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 of65ZnCl2 was added to this solution. For the equilibration

of the zinc solution containing 65Zn with other compo-

nents of the medium, the mixture was incubated at 25 �Cfor 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 · 104Æ250 lL)1), and incubated for 10 min at 37 �C.Then, the cells were mixed with the same volume of

uptake buffer containing 65ZnCl2 (the final concentration

of ZnCl2 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-counterARC-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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SLC39A14, a LZT protein, is induced in adipogenesis and transports zinc

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