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

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

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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: 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. 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 (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 [ 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). 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Ltd), a SLC3 9A1 4-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¢. functions of SLA3 9A1 4 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

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