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,3
* Departments of Medicine and Pediatrics and the Howard Hughes Medical Institute, University of California, San Francisco, CA, and Department of Biochemistry and Molecular Biology, University of Minnesota Medical School, Duluth, MN
3 To whom correspondence should be addressed. E-mail: jprohask{at}d.umn.edu.
ABSTRACT
Studies were conducted to determine distribution of the copper transporter, Ctr1, a transmembrane protein responsible for cellular copper uptake, in adult mice and in suckling mice nursed by either copper-adequate (Cu+) or copper-deficient (Cu–) dams. Western immunoblot analyses, using immunopurified antibody, detected monomeric (23 kDa) and oligomeric forms of Ctr1 in the membrane fraction of several mouse organs. Immunohistochemical analyses detected abundant Ctr1 protein in liver canaliculi; kidney cortex tubules; small intestinal enterocytes; the choroid plexus and capillaries of brain; intercalated disks of heart; mature spermatozoa; epithelium of mammary ducts; and the pigment epithelium, outer limiting membrane, and outer plexiform layer of the retina. Duodenal Ctr1 distribution was different in the adult compared with suckling mice; adult mice demonstrated strong intracellular staining of the enterocyte, whereas apical staining predominated in suckling mice. In Cu– mice at postnatal d 16 (P16), Ctr1 staining was augmented in kidney, duodenum, and choroid plexus, compared with Cu+ mice. Brain immunoblot data indicated that Ctr1 protein in membrane fractions of Cu– mice was 56% higher than Cu+ mice. Cu– mice had lower hemoglobin (56% of Cu+), and lower copper concentration (% of Cu+) in liver (15%), brain (26%), and kidney (65%). These results suggest that Ctr1 protein is expressed in multiple tissues and found in higher levels in selected organs after perinatal copper deficiency. Enhanced Ctr1 levels and redistribution might compensate in part for the decrease in copper supply. Mechanisms for the enhancement in Ctr1 staining remain to be established.
KEY WORDS: • copper deficiency • mice • copper transport protein • Ctr1 distribution
All living organisms require copper for growth and development. This requirement is thought to be due to the role copper plays as a cofactor for proteins involved in a variety of biological reactions, such as photosynthesis, respiration, free radical eradication, connective tissue formation, iron metabolism, and neurological function. However, free copper in excess of cellular needs mediates free radical production and direct oxidation of lipids, proteins, and DNA. Consequently, intracellular copper content is maintained by evolutionarily conserved cellular transport systems that regulate uptake, export, and intracellular compartmentalization (1). In complex organisms such as mammals, the balance between copper necessity and toxicity is achieved at both the cellular and organ level (2). Copper homeostasis is thought to be maintained by adjusting copper absorption in the duodenum and copper excretion in hepatic biliary processing. Copper absorption in the enterocyte is thought to be controlled by the plasma membrane copper transport protein (Ctr1)4 and the copper efflux transporter ATP7A, which moves from the Golgi to the basal lateral membrane when transferring copper for export (3).
Previously, the human gene CTR1 (SLC31A1) was identified by complementation of a yeast mutant (ctr1) that is defective in high-affinity copper uptake and is required for copper uptake in eukaryotic cells (4,5). Transcripts for hCTR1 were detected in 16 different human tissues (5). A second homologous gene notated as hCTR2 was also detected. CTR1 was predicted to contain 190 amino acids and possess 3 transmembrane domains. A number of excellent cell culture studies have characterized the biochemistry of CTR1 to show that it has high affinity for and transports the cuprous ion specifically (6–8).
Mouse Ctr1 is 92% identical to hCTR1 and comprises a 188 amino acid protein (9). Transcripts for mCtr1 were detected in multiple mouse tissues and were especially abundant in liver, kidney, and testis. The abundance of Ctr1 mRNA was not affected by dietary copper deficiency in rat brain, liver, or intestine (9). Two groups independently and coincidentally provided strong genetic evidence for the essential role of Ctr1 in mouse development (10,11). Early embryonic lethality was associated with Ctr1 deletion. In addition, mRNA expression studies using in situ hybridization also strongly suggested a ubiquitous role for Ctr1 in copper uptake in developing and adult mice, coupled with a more specialized role in epithelial cells and connective tissue (10).
Isotopic tracer studies of copper metabolism in rats suggest that intestinal uptake of copper is higher during copper deficiency (12). Studies in humans also suggest that copper uptake is regulated by dietary copper (13). Could this be due to alterations in Ctr1 expression? The purpose of the current studies was to characterize the tissue distribution of Ctr1 protein in normal adult mice using immunohistochemical techniques and to determine the effect of late maternal gestational and lactational dietary copper deficiency on Ctr1 expression in offspring.
METHODS AND MATERIALS
Animal care and diets. Copper deficiency was studied at the University of Minnesota Duluth (UMD). Adult male and female Hsd:ICR (CD-1) outbred albino mice were purchased commercially (Harlan Sprague Dawley). Dams received 1 of 2 dietary treatments, copper-deficient (Cu–) or copper-adequate (Cu+), consisting of a copper-deficient purified diet (Teklad Laboratories) and either low-copper drinking water or copper-supplemented drinking water, respectively. The purified diet was similar to the AIN-76A diet (14,15). It contained the following major components (g/kg diet): sucrose, 500; casein, 200; cornstarch, 150; corn oil, 50; cellulose, 50; modified AIN-76 mineral mix, 35; AIN-76A vitamin mix, 10; DL-methionine, 3; choline bitartrate, 2; and ethoxyquin, 0.01. Cupric carbonate was omitted from the AIN-76 mineral mix. The purified diet contained 0.33 mg Cu/kg and 44 mg Fe/kg by chemical analysis. Dams administered the Cu– treatment drank deionized water, whereas Cu+ treatment groups drank water that contained 20 mg Cu/L through the addition of CuSO4. Mice had free access to diet and drinking water. All mice were maintained at 24°C with 55% relative humidity with a 12-h light cycle (0700–1900). All protocols were approved formally by the University of Minnesota Animal Care Committee.
Previous experience with this mouse strain suggested a mean gestation period of 20 d. Dietary treatment of dams began on embryonic d 16 (E16). On postnatal d 2 (P2), the litter size was adjusted to 10 pups/dam. Pups were killed at P16 and P18 because signs of severe copper deficiency were evident. A total of 8 litters (4 Cu+ and 4 Cu–) were sampled. This paradigm is similar to that described previously (16).
Several organs were examined from adult, P120, Hsd:ICR male mice fed a copper-adequate nonpurified diet, Purina Laboratory Rodent Chow 5001, to prepare membranes for Western blots. Enterocytes were isolated from 10 cm of small intestine using EDTA treatment (17).
Adult, 10-wk-old, female and male mice of the same strain consuming a copper-adequate commercial diet were killed and tissues processed for immunohistochemistry at the University of California San Francisco (UCSF) following approved protocols. Mice were purchased from Charles River and fed a radiated PicoLab 5058 commercial diet.
At UMD, 1 male P16 mouse from each litter was anesthetized with diethyl ether and decapitated. A sample of blood was collected to measure hemoglobin. A portion of liver, 1 kidney, duodenum (first 5 cm of small intestine from the pyloric sphincter), half brain, right gastrocnemius muscle, lung, heart, testes, and eye were removed and placed in Bouin's solution (Sigma Chemical) overnight. Tissues were rinsed with deionized water and washed in 70% ethanol. They were then shipped to UCSF in 70% ethanol. The rest of the liver, half brain, and remaining kidney were processed for metal analysis for each mouse. Additional male pups (n = 8; P18) were killed and whole brains were processed for immunoblots.
Biochemical analyses.
Plasma was used to measure ceruloplasmin activity by following the oxidation of o-dianisidine (18). Portions of the diet (
1 g), and portions of liver, brain, and kidney tissue were weighed to the nearest 0.1 mg and wet-digested with 4 mL of concentrated HNO3 (Trace Metal grade, Fisher Scientific), and the residue was dissolved in 0.1 mol/L HNO3. Samples were then analyzed for total copper by flame atomic absorption spectroscopy (Model 1100B, Perkin-Elmer). Protein homogenates from other tissues of littermates were used for immunoblots and total protein estimated using a modified Lowry method with bovine albumin as a standard (19).
Western immunoblots. Western blotting analysis was performed by size fractionation of proteins on 12% SDS-PAGE gels and electroblot transfer to 0.2 µmol/L nitrocellulose membranes (Protran). Membranes were stained with Ponceau S (Sigma Chemical) to verify equal loading and transfer of protein and then were used in immunoblotting as described elsewhere (20). Some membranes were reprobed after incubation of membranes with buffer containing 2-mercaptoethanol and SDS at 55°C for 30 min. Selected immunoblots were reprobed for glucose transporter 1 (GLUT1) as described recently (21).
Ctr1 protein was detected using affinity purified rabbit anti-Ctr1 antibody. The antiserum was raised in rabbits against the following peptide: NH2-CRKSQVSIRYNSMPVPGPNGT-COOH corresponding to amino acids 95–114 of CTR1 with a terminal coupling cysteine added (Genemed Synthesis). This amino acid sequence is identical in mouse Ctr1. Immune serum was affinity purified over thiol-linked antigen coupled columns according to the manufacture's guidelines (Sulfo Link Pierce Chemical). In blocking experiments, anti-Ctr1 purified antibody was diluted 20-fold in PBS with at least 10–5 mol/L blocking peptide and incubated at 37°C for 3 h before being adding to membrane incubations. Experiments with preimmune serum consisted of incubations with serum diluted 1:500.
Specific secondary antibodies were diluted 1:10,000. SuperSignal West Pico chemiluminescent substrate (Pierce) was used to detect selected proteins. Chemiluminescence was captured using high-speed blue X-ray2 film (Lake Superior X Ray) and densitometry was carried out using the FluorChemTM system (Alpha Innotech). The size of the immunoreactive bands was estimated from regression analysis using standard peptides (Bio-Rad).
Membrane extracts for Ctr1 determination were prepared from brain, liver, kidney, and small intestine enterocyte tissues using a modified protocol (22). Briefly, tissues were homogenized with 15 volumes of 0.02 mol/L Tris-HCl (pH 7.4) containing 0.04 mol/L NaCl and 0.001 mol/L dithiothreitol. Homogenates were centrifuged at 4°C, 300 x g for 5 min and the supernatant was centrifuged for 30 min at 100,000 x g. The soluble fraction was saved and the membrane pellet solubilized in a buffer containing 0.06 mol/L Tris-HCl (pH 6.8), 50 g/L SDS, and 100 g/L glycerol. Aliquots of protein were boiled in loading buffer containing 20 g/L SDS and 0.14 mol/L 2-mercaptoethanol.
Immunohistochemistry. Various tissues from ICR (CD-1) 10-wk-old mice were isolated and fixed for 12–14 h in Bouin's fixative (Sigma Chemical). Tissues from P16 mice were fixed in Bouin's at UMD and received at UCSF. Organs were washed in 70% ethanol, dehydrated, embedded in paraffin, and sectioned (8 µm) as previously described (23). Paraffin sections were dewaxed, rehydrated, steamed for 30 min in 0.001 mol/L EDTA followed by treatment with 50 g/L hydrogen peroxide before staining. Sections were immunostained using standard procedures with the affinity-purified anti-Ctr1 at 1:200 dilution and enhanced with ABC Elite Vector Stain Substrate Kit (Vector Laboratories) using the manufacturer's protocol as previously described (24). Staining was visualized with 3,3'-diaminobenzidine (DAB substrate Kit; Vector Laboratories) and counterstained with Nuclear Fast Red (Vector Laboratories) including the nickel solution. Controls included tissues incubated with preimmune serum or anti-Ctr1 serum preadsorbed with 10–5 mol/L immunizing peptide for 48 h at 4°C. Sections were examined using a Nikon E800 Eclipse microscope and images captured using a Spot II digital camera.
Statistics.
Dietary treatment effects were evaluated by Student's t test after variance equality was tested, 
= 0.05. Data were analyzed using a personal computer and statistical software (Statview 4.5, Abacus Concepts).
RESULTS
Ctr1 Western analyses.
Membrane extracts from several 4-mo-old male mice were analyzed for Ctr1 expression (Fig. 1). Although the predicted monomeric size for mCtr1, containing 188 amino acids, is 24 kDa, several additional immunoreactive bands were detected after reducing and denaturing PAGE. The size estimates of these specific bands were consistent with homodimer and homotrimer formation. Adsorbed antibody by preincubation with an excess of antigen peptide blocked detection of most bands, including these predicted oligomeric forms of Ctr1 (Fig. 1A), thus confirming the specificity of the anti-Ctr1 antibody. Interestingly, the banding pattern and intensity was tissue specific (Fig. 1B). Adult mouse heart and brain contained larger bands. Detection of Ctr1 oligomers was not appreciably altered by increasing mercaptoethanol, by boiling the samples before loading, or by treatment with PNGase F (New England Biolabs) before electrophoresis.
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DISCUSSION
The results of the current studies indicate that Ctr1 protein is widely expressed in mammalian tissues in selected cell types. In general, these novel protein data support earlier in situ hybridization studies that evaluated Ctr1 mRNA expression (10). The protein expression of Ctr1 is somewhat age dependent; for example, P16 mice and 10-wk-old mice differed in the expression of Ctr1 in the duodenum, choroid plexus, and testes. In addition, the physiological state of a tissue can affect Ctr1 expression. Mammary tissue during pregnancy and lactation displayed more intense Ctr1 staining than involuted mammary tissue or staining in virgins. Others detected Ctr1 staining in rat mammary tissue using an antiserum developed against a similar sequence of CTR1 (amino acids 90–110) (25). Perhaps the high abundance of Ctr1 in mammary tissue has to do with the remarkable diversion of copper to this organ during pregnancy in preparation for lactation (26).
Data in the current studies suggest that Ctr1 protein expression is higher in duodenum, kidney, and brain of copper-deficient mice, whereas other tissues did not display this pattern robustly. Teleologically, it makes sense for the enterocyte to increase Ctr1 during times of copper limitation and to decrease Ctr1 upon exposure to high luminal copper. Tracer studies mentioned previously support this proposition (12,13).
Similarly, the kidney might be expected to upregulate Ctr1 levels to retain copper when limiting in the blood. Staining in the kidney tubules clearly is consistent with Ctr1 working to pump glomerular filtrate copper back into blood. High expression of Ctr1 in the choroid plexus and renal tubules is noteworthy, given their parallel functions.
Perhaps the upregulation of Ctr1 protein in the choroid plexus of copper-deficient mice is an attempt to increase copper uptake into brain by restricting copper in cerebrospinal fluid (CSF). Staining on the apical epithelial membrane is consistent with increased capacity of Ctr1 to extract copper from CSF. The apical and basolateral membranes of the choroid plexus epithelia express many cation and anion pumps in a heterogenous pattern (27). It is interesting to note that the expression of the divalent metal transporter (DMT)1, thought to pump ferrous iron, is also lower in choroid plexus of suckling rats (P18) compared with adults (28). Furthermore, DMT1 staining of rat choroid plexus epithelial cells revealed a punctate distribution in the cytoplasm, clearly different from Ctr1 in mouse choroid plexus.
It was somewhat surprising that liver did not show an upregulation of Ctr1 in copper-deficient mice. Work in rats indicated an enhanced capacity of hepatocytes from copper-deficient rats to transport labeled copper compared with controls (29). However, the liver of rats is more vulnerable to copper deficiency than that of mice (18).
Our immunoblot data on P16 mice suggest that more total Ctr1 protein is present in brain membrane extracts after copper limitation. The immunohistochemical data indicated that the choroid plexus was especially enriched in Cu– mice; thus, further characterization of this tissue after copper deficiency will be important.
Although Ctr1 protein expression was not studied previously in mammalian tissues derived from copper-deficient animals, there are some previous studies that are pertinent and somewhat puzzling. Copper supplementation studies in rat pups treated orally with copper reported an increase in Ctr1 protein in intestine (30). In a similar experiment, Ctr1 protein levels (at least for 1 band shown) were higher for intestine and liver of copper-treated pups (31). Interestingly, the higher intestinal levels of Ctr1 protein of P20 copper-treated rats occurred with no increase in intestinal copper concentration (31). Further work remains to determine the mechanism for changes in Ctr1 protein in response to changes in copper homeostasis in mammals.
Expression data on Ctr1 from the current studies show the importance of cellular location, how it changes with age, and perhaps in response to copper availability. Some have suggested that the localization of Ctr1 between plasma membrane and an intracellular vesicular compartment depends on the cell type (7). Furthermore, others using transfected cell culture models are debating whether Ctr1 moves from the plasma membrane to an internal vesicular compartment when copper is present outside cells (32,33). In essence, if Ctr1 moved inside when copper was elevated outside, this would be a way to regulate copper transport. The increased enterocyte Ctr1staining we observed appeared to be on the apical surface with no evidence for translocation because this apical pattern was seen in the control age-matched pups.
One of the interesting issues concerning evaluation of Ctr1 protein expression is the appearance of homomultimers of this protein. The purified antibody used in the present studies recognized Ctr1 consistent with the monomer, dimer, and trimer forms of native mCtr1 in membrane preparations from several tissues. This occurred even after reducing SDS-PAGE with a generous supply of 2-mercaptoethanol and removal of carbohydrate from asparagine-15. The oligomeric bands were not present when the antibody was pretreated with excess antigenic peptide. Previous elegant work by others in cell culture also reported the presence of homomultimers of Ctr1 and the dependence on cysteine residues of this distribution (6,8). There are also sequences in the third transmembrane domain that affect oligomerization (34). Further experiments will be required to characterize the oligimerization and expression of this essential copper transport protein.
ACKNOWLEDGMENTS
We appreciate the excellent technical assistance of Margaret Broderius and Bruce Brokate (UMD) and Ivy Hsieh, Haidong Yang, Mark Sternlicht, and Philip Ursell (UCSF).
FOOTNOTES
1 Supported by grant HD-39708 (J.P.) from the National Institutes of Health. 
2 Investigator with the Howard Hughes Medical Institute.
4 Abbreviations used: CSF, cerebrospinal fluid; Ctr1, copper transport protein; Cu+, copper-adequate; Cu–, copper-deficient; DMT, divalent metal transporter; E, embryonic day; GLUT1, glucose transporter 1; P, postnatal day; UCSF, University of California San Francisco; UMD, University of Minnesota Duluth.
Manuscript received 30 August 2005. Initial review completed 14 October 2005. Revision accepted 27 October 2005.
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