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Metallomics Accepted Manuscript

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DOI: 10.1039/C5MT00097A

Copper uptake by DMT1: A compensatory mechanism for CTR1 deficiency in human umbilical vein endothelial cells

Chen Lin,a,* Zhen Zhang,a,b,* Tao Wang,a Chen Chen,a and Y. James Kanga,c,# a

Regenerative Medicine Research Center and bState Key Laboratory of Biotherapy, West China

Hospital, Sichuan University, Chengdu, Sichuan, P. R. China; cDepartment of Pharmacology and Toxicology, University of Louisville, Louisville, Kentucky, USA

Running title: CTR1 and DMT1 for copper uptake *CL and ZZ made equal contributions to this study. #Correspondence

address:

Dr. Y. James Kang Regenerative Medicine Research Center West China Hospital, Sichuan University Chengdu, Sichuan 610041, P. R. China Telephone: (86) 028-8516-4037 Fax: (86) 028-8516-4037 E-mail: [email protected]

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Abstract Copper transport 1 (CTR1) plays a critical role in copper uptake by cells, but several studies demonstrated that divalent metal transporter 1 (DMT1) also transports copper in some cells and under certain circumstances. The present study was undertaken to determine the relationship between CTR1 and DMT1 in copper uptake. Human umbilical vein endothelial cells (HUVECs) were exposed to increasing concentrations of extracellular copper in cultures, leading to increased accumulation of copper in cells proportional to concentrations of extracellular copper. However, CTR1 proteins decreased in relation to the increase in copper concentrations, and DMT1 increased inversely correlating to the decrease in CTR1. Gene silencing of either CTR1 or DMT1 did not affect copper accumulation in cells, but deficiency in both CTR1 and DMT1 resulted in a complete inhibition of copper uptake. This study thus demonstrates that DMT1 imports copper under the condition of CTR1 deficiency, and vice versa. Therefore, CTR1 and DMT1 would compensate for each other for copper uptake in mammalian cells, although different types of cells may use either one as a predominant copper importer under physiological conditions.

Keywords: Copper, CTR1, DMT1, uptake, endothelial cells

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Introduction Copper is an essential trace element in humans, but it is also potentially toxic because of the ability of the metal ions to catalyze the formation of free radicals.1 Thus, copper is tightly regulated for its homeostasis in the mammalian system. The cellular and organismal levels of copper are equilibrated through the regulation of the entry and exit mechanisms as well as the processes whereby copper is delivered to specific subcellular locations.2, 3 A number of proteins involved in copper uptake by, distribution in, and export from the cells have been identified,4-11 providing a fundamental understanding of copper homeostasis. A major transporter mediating copper entry into cells is the copper transporter 1 (CTR1), a glycosylated trans-membrane protein.12,13 CTR1 was first discovered as a protein responsible for high-affinity copper uptake in yeast about 20 years ago,6 and then verified to be the primary copper transporter in mammals.8 CTR1 knockout mice exhibits tissue-specific defects in copper accumulation and embryonic lethality,14,15 indicating a crucial role for copper acquisition through CTR1 for mammalian function and embryonic development. In addition to CTR1, its homologs such as CTR2 have been identified to selectively function in some organs and under certain circumstances.16, 17 However, copper transportation across cell membrane remains active in CTR-deficient cells,18 suggesting that CTR may not be the only transport system involved in copper uptake in mammalian system. Divalent metal transporter 1 (DMT1), a relatively nonspecific divalent metal ion transporter, is believed to play a role in copper uptake in mammalian system, such as in the small intestine.11,19 In iron-GH¿FLHQW UDWV DMT1 enhances copper uptake into duodenal enterocytes.20 In 3

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addition, in iron-deprived human embryonic kidney cell line (HEK-29) DMT1 becomes a major copper transporter.20 Therefore, in specific organ systems or under certain circumstances, DMT1 is or becomes an important copper transporter. However, it is critical to know how DMT1 correlates with CTR1 in this process. The present study was undertaken to specifically address the relationship between CTR1 and DMT1 in copper uptake in human umbilical vein endothelial cells (HUVECs). We examined alterations in CTR1 and DMT1 under conditions of varying extracellular copper concentrations, and defined the condition under which DMT1 became dominant in copper uptake. The results demonstrate that DMT1 plays a compensatory role in copper uptake for CTR1 deficiency in the HUVECs.

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Materials and Methods Cell culture and treatment HUVECs acquired from American Tissue Culture Collection (ATCC, Virginia, USA) were maintained at 37 ºC in L-DMEM (GIBCO, USA) supplemented with 10 % fetal bovine serum (FBS, Hyclone, USA) and 1 % penicillin/streptomycin (GIBCO, USA) in 5 % CO2 incubator. Cells were passaged by 0.25 % trypsin (GIBCO, USA) and 1 % EDTA in Ca2+- and Mg2+-free phosphate-buffered saline (PBS). Experimental cells were subcultured in six-well plates at a density of 2×105 cells overnight. To determine the effect of copper sulfide on CTR1 or DMT1, cells were treated with varying concentrations of CuSO4 (Sigma, USA) in FBS-free L-DMEM (copper content less than 0.1 M) when the monolayer cell cultures reached about 50 % confluence and lasted for 24 h. To examine whether the deficiency in CTR1 or DMT1, or both in the cells have any impacts on copper transport capacity, cells were transfected with corresponding siRNA for 24 h and then incubated with FBS-free media containing 5 M CuSO4 for another 24 h. CuSO4 was dissolved in deionized water and sterilized using a filter before they were added to the cells. Gene silencing of CTR1 and DMT-1 The siRNA targeting human CTR1 or DMT-1 and negative mismatch siRNA were designed and synthesized from RiboBio (RiboBio, CN). The siRNA sequences for CTR1 wHUH GGAAGAAGGCAGUGGUAGU dTdT- ¶ siRNA sequences for DMT- ZHUH

¶-

¶-dTdT CCUUCUUCCGUCACCAUCA- ¶ 7KH

¶-CCUGGAUCCAGGAAAUAUU dTdT- ¶

¶-dTdT

GGACCUAGGUCCUUUAUAA- ¶ 7KH RSWLPDO WUDQVIHFWLRQ HIILFLHQF\ ZDV DVFHUWDLQHG IURP 5

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our preliminary study testing the range from 10-100 nM, and a condition of 70 %-80 % silencing effect with minimal cytotoxicity was selected for this study: 25 nM annealed siRNAs targeting human CTR1or DMT1 or negative mismatched siRNA in serum-free and antibiotics-free media. Lipofectamine RNA Imax (Invitrogen, USA) was used as the transfection reagent following the manufacturer's instruction. The optimal transfection efficiency was 25 nM Lipofectamine RNA Imax. After 24 h siRNA transfection, cells were cultured in FBS-free L-DMEM or the media added

0 &X624 for another 24 h and then harvested for further analysis.

Copper concentration determination Intracellular copper concentration was determined with graphite furnace atomic absorption spectrophotometer (Thermo, USA). Briefly, cells were collected by cell scraper, washed tree times with ice-cold PBS containing 10 mM EDTA (Sigma, USA) to ensure that extracellular copper was completely removed, followed by centrifugation at 1,500 rpm for 5 min. Cell pellets were lysed with 1 % SDS solution (Beyotime, CN) and ultra-sonication. Lysates were divided into two parts. One was digested with concentrated nitric acid at 50 ºC for 72 h and analyzed using a graphite furnace atomic absorption spectrophotometer. Another was used for the determination of protein concentrations by the DC Protein Assay (Bio-Rad, USA). Intracellular copper concentrations were normalized by total protein concentrations. Western blotting analysis of proteins The protein levels of CTR1 or DMT1 were determined by Western blotting. Briefly, cells were collected by cell scraper, washed tree times with PBS and centrifuged at 1,500 rpm for 5 min. Protein extracts were acquired after lysing cells in 1% SDS solution (Beyotime, CN) 6

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containing 1 % complete EDTA-free protease inhibitor cocktail (Roche, DE). After ultrasonication and determination of protein concentration, protein samples were mixed with 5× loading buffer, boiled for 10 min at 100 ºC and cooled. An aliquot of protein (30 J IURP HDFK sample was separated by 10 % SDS-PAGE. Proteins were then transferred onto a polyvinylidene difluoride membrane (Bio-Rad, USA). Membranes were blocked with 5 % nonfat dry milk in Tris-HCl buffer solution containing Tris-HCl (10 mM, PH 8.0), NaCl (150 mM) and Tween-20 (0.1 %) (TBS-T) for 1 h at room temperature. The blots were incubated overnight at 4 ºC with respective primary antibodies (anti-CTR1, ab129067, Abcam, USA; anti-DMT1, sc-166884, Santa Cruz, USA; anti-CCS-1, sc-20141, Santa Cruz, USA; anti- -actin, ab8226, Abcam, USA) in blocking solution according to the YHQGHU¶V recommendation. After incubation, the blots were washed with TBS-T six times for 5 min each followed by incubation with respective secondary antibody for 1 h at room temperature. After washing six times (5 min each), target proteins were visualized using chemiluminescence HRP substrate (Millipore, USA) and analyzed by densitometry using Image Pro Plus Software. Real-time quantitative RT-PCR Total RNA was extracted from HUVECs with TRIzol reagent (TaKaRa, JP) according to the manufacturer's instruction. RNA concentration was quantified with Ultraviolet spectrophotometer (Thermo, USA). Complementary DNAs (cDNAs) were synthesized with a SYBR Premix Ex Taq (TaKaRa, JP) in MJ Mini personal thermal Cycler (Bio-Rad, USA). The amount of cDNA corresponding to 100 ng of RNA was amplified using a SYBR green PCR kit (Applied Biosystems) with the primers for CTR1, DMT1 or -actin. The primer sequences 7

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(Table 1) were designed using the software offered by Takara. Real-time RT-PCR reactions were performed, recorded, and analyzed by the iCycler. Lactate dehydrogenase (LDH) release assay LDH release assay was used to detect the changes in cell membrane permeability after siRNA or copper treatment. Lactate dehydrogenase activity in cells or cultured media were measured spectrophotometrically at 450 nm with LDH Assay Kit (Nanjing Jiancheng %LRWHFKQRORJ\ ,QVWLWXWH &1 UHVSHFWLYHO\ DFFRUGLQJ WR WKH PDQXIDFWXUHU¶V LQVWUXFWLRQV In this assay, LDH catalyzed the reduction of NAD to NADH, then INT (2-p-iodophenyl-3-nitrophenyl tetrazolium chloride) was reduced to red formazan by diaphorase. The absorbance or OD at 450 nm was regarded as relative content of LDH. LDH release ratio was calculated as follows equation. LDH release ratio = LDH medium / (LDH medium + LDH cell) Statistics Data were obtained from more than three separate experiments and presented as mean values ± S.E.M. One-ZD\ $129$ ZDV XVHG IRU LQLWLDO DQDO\VLV DQG 6WXGHQW¶V t-test was employed for comparison among multiple groups, and P < 0.05 was consider significant at differences among treatments.

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Results Changes in CTR1 and DMT1 in response to varying concentrations of extracellular copper Copper accumulation in HUVECs in response to varying concentrations of extracellular copper was determined, as shown in Fig 1A. The increase in copper accumulation in the cells was positively correlated with the increase in concentrations of extracellular copper. Under the condition of varying concentrations of extracellular copper, changes in CTR1 and DMT1 protein levels were examined. The results shown in Fig 1B and C revealed that CTR1 protein levels decreased inversely correlating to the increase in concentrations of extracellular copper, but DMT1 protein levels were progressively increased as the increase in copper concentrations. To determine whether the changes in copper transporter protein levels resulted from alterations in gene expression, the levels of mRNA for CTR1 or DMT1 were determined as a function of varying copper concentrations. As shown in Fig 1D, there were no significant changes in the mRNA levels for either CTR1 or DMT1. To further probe possible factors that affect the observed changes in CTR1 or DMT1, changes in intracellular copper chaperone for superoxide dismutase-1 (CCS-1) protein levels were examined. As shown in Fig S1, under the same copper exposure condition, CCS-1 protein levels were not significantly altered. Compensation relationship between CTR1 and DMT1 To define the relationship between CTR1 and DMT1 in copper uptake in HUVECs, deficiency in CTR1 or DMT1, or both in the cells was made by siRNA targeting the proteins. Gene silencing of CTR1 in HUVECs dramatically decreased both the mRNA and protein levels of CTR1 (Fig 2A), and the same result for DMT1 was also obtained (Fig 2B). In addition, the 9

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inverse correlation between CTR1 and DMT1 was also observed under the deficiency in either transport protein. The results shown in Fig 2C revealed that DMT1 protein levels were significantly increased in CTR1 deficient cells and vice versa, while the addition of copper did not cause further changes. The cells deficient in CTR1 or DMT1, or both were exposed to 5 PM copper in culture media, and copper accumulation in these cells after 24 h exposure was determined. As shown in Fig 2D, copper accumulation in the cells deficient either CTR1 or DMT1 was not affected in comparison with either untreated control or mismatch siRNA-treated cells, but in the cells deficient in both CTR1 and DMT1 copper accumulation was completely suppressed. Effects of CTR1 and DMT1 deficiency on cell membrane permeability To eliminate the impact of possible changes in cell membrane permeability on intracellular copper accumulation, the effect of deficiency in CTR1 or DMT1, or both on LDH release in HUVECs was determined. The results shown in Fig 3 demonstrated that neither copper treatment nor gene silencing of CTR1 or DMT1, or both, increased cell membrane permeability in comparison with untreated controls.

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Discussion CTR1 is a major transporter mediating copper entry into mammalian cells for cellular function and provides an essential support for mammalian embryonic development.14,15 However, recent studies demonstrated a critical role of DMT1 in copper uptake by intestinal cells.20 This may also takes place in other types of cells. If DMT1 represents another major copper import system in mammalian cells, an important question is how the two copper transporters are coordinated under varying conditions. The answer obtained from the present study is that under the condition of increasing elevation of extracellular copper concentrations, a gradual depression of CTR1 takes place, and DMT1 compensates for CTR1 deficiency in copper uptake; and under the deficiency in either transporter condition, one plays a compensatory role for the other in copper accumulation. It would be expected to observe that with the increase in concentrations of extracellular copper, CTR1 would enhance its activity for copper transportation. In the present study, it was surprisingly observed that with the increase in concentrations of extracellular copper, CTR1 protein levels actually decreased inversely correlating to the increase in copper concentrations. The decrease in CTR1 protein levels in response to the increase in copper concentrations was also observed previously.21,22 An important finding in the present study was that the reduction in CTR1 protein levels did not cause suppression of copper accumulation in the cells. It appeared that DMT1 would be responsible for the unhalted copper accumulation in the cells because DMT1 protein levels increased as a function of the increase in concentrations of extracellular copper. The inversely-correlated changes in CTR1 and DMT1 protein levels in response to the 11

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increase in concentrations of extracellular copper would not result from alterations in gene expression because there were no changes in mRNA levels for either CTR1 or DMT1. In addition, CCS-1, a major intracellular copper chaperone, did not change in its protein levels under the same copper exposure condition, suggesting intracellular trafficking may neither be affected by nor impact on the changes in CTR1 or DMT1. It will be our future undertaking to examine how the changes in CTR1 and DMT1 take place. DMT1, a polytopic membrane protein composed of 12 transmembrane segments,23 is expressed at the plasma membrane and late endosomes,24, 25 allowing for the uptake of nonprotein-bound metals from the extracellular environment and/or the recycling endosomes.26, 27 It is considered to play a central role in the regulation of membrane transportation of iron as well as other metals via a proton-coupled mechanism.7,28 An early study demonstrated that DMT1 is a physiologically relevant cuprous copper transporter in intestinal cells.11 However, there is a controversy about whether DMT1 transports copper.29 Recent studies in iron-GH¿FLHQW rats and iron-deprived HEK-293 cells showed that DMT1 participates in copper import during iron GH¿FLHQF\ 20 In the present study, it was observed that DMT1 became increasingly important in copper accumulation under the condition of copper overload. How does DMT1 correlate with CTR1 for copper transportation? That increased DMT1 protein levels were associated with decreased CTR1 levels or vice versa, as shown under the condition of either copper exposure or deficiency in either protein, suggest there is compensatory relationship between CTR1 and DMT1. This was confirmed by the result that deficiency in either CTR1 or DMT1 did not affect copper accumulation, but deficiency in both CTR1 and DMT1 12

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completely blocked copper accumulation. However, it appeared that under physiological condition, CTR1 would play a dominant role in copper accumulation in the HUVECs. Only when CTR1 activity decreases, does DMT1 become predominant, compensating for the deficiency in CTR1 activity. Why does the increase in extracellular copper concentrations suppress CTR1 protein levels? An early report showed that a short time copper treatment (less than 2 h) in HEK293 stimulated the endocytosis and degradation of CTR1 in a dose-dependent manner.21 Further studies explained that the copper-dependent endocytosis of CTR1 was an acute and reversible mechanism for the regulation of cellular copper entry.22 However, in the present study, the exposure of HUVECs to increasing concentrations of extracellular copper lasted for 24 h and the decrease in CTR1 protein levels was measured after the exposure. It thus may suggest that CTR1 might undergo degradation process under current experimental condition. A previous study in the Caco-2 TC7 cell model of human intestinal epithelial cells has demonstrated that DMT1 protein and mRNA levels were decreased following exposure to 100 M copper for 24 h.30 It is possible that the predominant copper importer in intestinal cells under physiological conditions is DMT1 as it is highly expressed in these cells. Thus, it is also possible that with the increase in copper concentrations, CTR1 may increase along with DMT1 decreases, but this was not examined in previous studies. It is important to understand the mechanism by which the inverse changes in CTR1 and DMT1 take place in response to increasing concentrations of extracellular copper. This is a limitation of the present study, but it needs a comprehensive effort in the future to identify 13

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molecules involved in this process. In summary, the present study produced data that show under the condition of increasing concentrations of extracellular copper, the primary copper transport CTR1 in the HUVECs gradually declines most likely due to its internalization and degradation process, and the DMT1 gradually increases to compensate for the loss of CTR1 in copper transportation. Only under the condition of deficiency in both CTR1 and DMT1 is copper import affected. Therefore, the evidence obtained here demonstrates a mutual compensatory relationship between DMT1 and CTR1 for copper accumulation in mammalian cells.

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Acknowledgments The authors thank Xiaoming Hou and Suzhen Fan for technical support. This work was supported by National Science Foundation of China (grant number: 81230004 to Y. J. Kang). The funding sources had no influence in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the article for publication.

Author Contributions All authors participated in the design, interpretation of the studies, analysis of the data, and review of the manuscript; LC, ZZ, TW and CC carried out the experiments; YJK and TW wrote the manuscript.

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DOI: 10.1039/C5MT00097A

28. M. D. Fleming, C. C. Trenor, M. A. Su, D. Foernzler, D. R. Beier, W. F. Dietrich and N. C. Andrews, Nat. Genet., 1997, 16, 383-386. 29. M. Knöpfel, C. Smith and M. Solioz, Biochem. Bioph. Res. Co., 2005, 330, 645-652. 30. J. Tennant, M. Stansfield, S. Yamaji, S. K. Srai and P. Sharp, FEBS Lett., 2002, 527, 239-

Metallomics Accepted Manuscript

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Metallomics View Article Online

DOI: 10.1039/C5MT00097A

Figure Legend Fig 1. Effects of extracellular copper on CTR1 and DMT1. (A) Changes in intracellular copper concentrations after copper exposure. (B) Western blotting analysis of changes in CTR1 and DMT1 proteins in response to copper exposure. (C) Semi-quantitative analysis of CTR1 and DMT1 protein levels detected by Western blotting. (D) Effects of copper exposure on mRNA levels of CTR1 and DMT1 detected by a real-time RT-PCR analysis. All of the data were collected from three independent experiments and expressed as mean ± S.E.M. Different labels indicate a significant di¡erent from each other (p < 0.05). Fig 2. Changes in copper accumulation in cells treated with siRNA targeting CTR1 or DMT1, or both. (A) and (B) The detection of the efficacy of gene silencing of CTR1 or DMT1 by RT-PCR and Western Blotting. (C) Western blotting analysis of changes in CTR1 or DMT1 proteins in response to siRNA targeting DMT1 or CTR1, respectively. (D) Changes in copper accumulation under different conditions. All of the data were collected from five independent experiments and expressed as mean ± S.E.M. *Significantly di¡erent from untreated controls (p < 0.05). Fig 3. Effects of siRNA or copper exposure on LDH release. All of the data were collected from three independent experiments and expressed as mean ± S.E.M.

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Metallomics Accepted Manuscript

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Metallomics

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DOI: 10.1039/C5MT00097A

Table 1 Primer sequences used for real-time quantitative RT-PCR analysis CTR1 DMT1 ACTIN

5'-CTGCTGCGTAAGTCACAAGTCAG-3' 3'-TATGACCACCTGGATGATGTGC-5' 5'-TTGCTGTCTTCCAAGATGTAGAG-3' 3'-GAGGATGGGTATGAGAGCAAAG-5' ¶-CCACGAAACTACCTTCAACTCC- ¶ ¶-GTGATCTCCTTCTGCATCCTGT - ¶

(forward) (reverse) (forward) (reverse) (forward) (reverse)

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DOI: 10.1039/C5MT00097A

Metallomics Accepted Manuscript

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)LJ

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DOI: 10.1039/C5MT00097A

Metallomics Accepted Manuscript

Metallomics

)LJ

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Metallomics Accepted Manuscript

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Copper uptake by DMT1: a compensatory mechanism for CTR1 deficiency in human umbilical vein endothelial cells.

Copper transport 1 (CTR1) plays a critical role in copper uptake by cells, but several studies demonstrated that divalent metal transporter 1 (DMT1) a...
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