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 dominan
Trang 1and transports zinc
Kei 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: imagawa@phar.nagoya-cu.ac.jp
(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.
Trang 2As 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 fragfrag-ments 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 n¼ 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
Trang 3introns 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)
(RT-3)
stop ATG
(R-5')
(R-3')
SLC39A14
262
489 aa
3660
Mouse
SLC39A14
Transmembrane domain
489 aa Mouse SLC39A14
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.
Trang 4Tissue 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 0.5 1 2 3 6 12 24
28S 18S
hr
SLC39A14
β-actin
0 1 3 6 12 24 2 4 6 8
3T3-L1 40000
20000
0
0 1 3 6 12 24 2 4 6 8
3T3-F442A 9000
4500
0
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).
1200000
0
gr o
w th
gr o
wth arre
sted
arre sted
pr olif era ting
pr olif erating
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).
Trang 5functions 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 and65ZnCl2, 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
4C, 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 differentiiniti-ation [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 Hear
200000
100000
0
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.
Trang 630 25 20 15 10 5 0
60 50 40 30 20 10 0
Time (min)
30 20
10 0
250 200 150 100 50 0
[Zn] ( µM)
5 4 3 2 1 0
***
*
**
***
SLC39A14Contr
o l
SLC39A14
Cont
ro l
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 M65Zn 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.
Trang 7underlying 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
SLC39A14in 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
Trang 8Total 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 hGen-omepage (http://genGen-ome.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)1of 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)1of 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 50C for 2 min and at 95 C for 10 min, and then the PCR was conducted at 95C for 15 s and at
60C 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)1of collagenase type II (Sigma-Aldrich, Inc., St Louis, MO, USA) in KRB buffer, contain-ing 4% bovine serum albumin at 37C 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 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
Trang 9pEGFP-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· 106cells 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 CMV 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)1G418 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 the65Zn uptake assay
65Zn uptake assay
65
ZnCl2 (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 of
65ZnCl2 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 25C
for 24 h before the experiment The cells were grown to
25% confluence, harvested by centrifugation at 150 g for
3 min at 4C, and washed in cold uptake buffer The
cells were resuspended in the prewarmed uptake buffer
(5· 104
Æ250 lL)1), and incubated for 10 min at 37C
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-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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