Department of Nutrition, University of California, Davis, Davis, California
Submitted 23 September 2004 ; accepted in final form 29 December 2004
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
zinc transport; mammary gland; lactation
The lactating mammary gland is a highly specialized secretory organ that tightly coordinates the accumulation, production, and secretion of milk components in a vectorial manner (21). Initial differentiation of proliferating mammary epithelial cells to a fully functional, secretory cell type and galactopoiesis (maintenance of established lactation) are hormonally regulated (3) and require both continuous and successive episodic hormonal stimulation, primarily through prolactin signaling pathways in the mammary epithelial cell (22). During lactation, a substantial amount of Zn2+ is taken up by the mammary gland and secreted into milk (0.51 mg of Zn2+/day), facilitating the movement of almost twice the amount of Zn2+ that is transferred daily across the placenta to the fetus during pregnancy (18), demonstrating the extraordinary activity of mammary gland Zn2+ transport. The initial step in milk Zn2+ secretion is Zn2+ import from the maternal circulation into the mammary gland. We have previously characterized the expression of Zip3 in the mammary gland of the lactating rat (16), which suggests that Zip3 may play a specific role in mammary gland Zn2+ import and may thus regulate milk Zn2+ secretion.
HC11 cells, a clonal derivative of the mammary epithelial COMMA-1D cell line, express functional prolactin receptors and are an excellent model for studying the progression of mammary epithelial cell differentiation to a secreting cell type (3) and the regulation of mammary gland gene expression and milk protein secretion after lactogenic hormone exposure (4). In this study, we used HC11 cells to test the hypothesis that Zip3 plays a major role in facilitating Zn2+ uptake into both proliferating and secreting mammary epithelial cells. The results of the present study describe effects of reduced Zip3 expression on cellular Zn2+ uptake and document effects of prolactin on the regulation of Zn2+ transport in mammary epithelial cells.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Affinity purification of Zip3 antibody. Peptide antigen (2 mg; FRRERPPFIDLETFNAGSDAGSDSEYESPF-Cys) was produced and conjugated to an affinity column (Sulfolink purification kit; Pierce Biotechnology, Rockford, IL), and Zip3 antibody was affinity purified from rabbit antiserum according to the manufacturer's instructions as previously described (16).
Localization of Zip3 in HC11 cells. HC11 cells were seeded onto glass coverslips and cultured for 16 h in growth medium and treated with differentiation medium for up to 24 h where noted. Medium was aspirated, and cells were washed extensively with phosphate-buffered saline (PBS), fixed in phosphate-buffered paraformaldehyde (4%) for 30 min, again washed in PBS, and then permeabilized with Triton X-100 (0.4% in PBS) for 4 min. Nonspecific binding was blocked with 10% goat serum and 1% bovine serum albumin in PBS for 30 min, followed by incubation with affinity-purified antibody (0.5 µg/ml) for 60 min with rocking at room temperature. After being washed extensively with PBS-Tween-20 (PBST, 0.05%), primary antibody was detected using Alexa 488-conjugated goat anti-rabbit IgG (1 µg/ml; Molecular Probes, Eugene, OR) for 45 min at room temperature with rocking and shielded from light. Stained cells were washed extensively in PBST, and then coverslips were drained, mounted in ProLong (Molecular Probes), and sealed with nail polish. Zip3 was colocalized with the plasma membrane after incubation with 1 µM 1,1-dihexadecyl-3,3,3'3'-tetramethylindocarbocyanine perchlorate (DiI; Molecular Probes) for 2 min at 37°C followed by 15 min at 4°C, and colocalization with the endosomal compartment was identified after incubation with transferrin 546 (Molecular Probes) for 5 min at 37°C. Cells were fixed and stained as described above. Immunofluorescence imaging was performed using an Olympus BX50WI microscope with UPlanApo x100 magnification under an oil-immersion lens (NA, 1.35), and digital images were captured using the Bio-Rad Radiance 2100 confocal system with LaserSharp2000 software, version 4.1 (Bio-Rad, Hercules, CA).
Inhibition of protein synthesis with cycloheximide. Confluent HC11 cells were cultured in differentiation medium for 24 h and treated with cycloheximide (25 µM) in differentiation medium for 48 h to inhibit protein translation. The medium was aspirated, and cells were prepared for RNA or protein extraction as described below.
Quantification of Zip3 mRNA levels using real-time RT-PCR.
Total RNA was isolated from HC11 cells using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions and diluted to 1 µg/µl in RNAse-free water. RNA integrity was evaluated using electrophoresis through 2% agarose and ethidium bromide staining (Sigma). cDNA was generated from 1 µg of RNA using a reverse transcription kit (PerkinElmer Applied Biosystems, Foster City, CA) following the manufacturer's instructions, and the reaction was performed at 48°C for 30 min followed by 95°C for 5 min. Real-time PCR was performed using the cDNA reaction mixture (1.5 µl for GAPDH and 4 µl for Zip3) and an ABI 7900HT real-time thermocycler (PerkinElmer Applied Biosystems) coupled with SYBR Green technology (PerkinElmer Applied Biosystems) using gene-specific primers to mouse Zip3 (forward, 5'-AACAGCATGTCAGCTTCTCCTATG-3'; reverse, 5'-GGATCCCGCCTGCACTAA-3') and GAPDH (control gene: forward primer, 5'-TGCCAAGTATGATGACATCAACAAG-3'; reverse primer, 5'-AGCCCAGGATGCCCTTTAGT-3'). The primers were chosen using Primer Express software (PerkinElmer Applied Biosystems) and were purchased from Qiagen (Valencia, CA). The following cycling parameters were used: 50°C for 2 min, 95°C for 10 min, 40 cycles of 95°C for 15 s, and 60°C for 1 min, followed by 95°C for 15 s. The linearity of the dissociation curve was analyzed using ABI 7900HT software, and the mean cycle time of the linear part of the curve was designated as Ct. Each sample was analyzed in duplicate and normalized to GAPDH using the equation Ct = CtZip3 CtGAPDH. Preliminary experiments allowed us to determine that expression of GAPDH was not affected by our treatments in this model (data not shown); therefore, GAPDH was used as a normalization control. The fold change in Zip3 expression of differentiated or Zip3 knockdown cells (test) relative to nondifferentiated or mock transfected cells (control) was calculated using the equation 2(
Ct
), where
CtGENE = mean
CtZip3 control
CtZip3 of test. Values represent mean fold changes ± SD.
Determination of Zip3 protein levels using Western blot analysis. HC11 cells were scraped into ice-cold lysis buffer (20 mM HEPES, pH 7.4, 1 mM EDTA, and 250 mM sucrose containing protease inhibitors; Sigma) and sonicated twice for 10 s on ice. The postnuclear supernatant was isolated by centrifugation for 5 min at 5,000 g at 4°C, and protein concentration was determined using the Bradford protein assay (Bio-Rad). Cell protein (200 µg) was separated using SDS-PAGE (10% polyacrylamide) and transferred onto nitrocellulose membranes for 90 min at 350 mA. Nitrocellulose membranes were blocked for 45 min at room temperature in 5% nonfat milk in PBS with 0.1% Tween-20, incubated with affinity-purified Zip3 antibody (1 µg/ml) for 45 min at room temperature, and detected with donkey anti-rabbit IgG conjugated to horseradish peroxidase (HRP; Amersham Pharmacia Biotech, Piscataway, NJ). Protein bands were visualized using the SuperSignal Femto chemiluminescence detection system (Pierce Biotechnology) and exposed to autoradiographic film. Relative band density was quantified using the Chemi-doc Gel Quantification System (Bio-Rad).
Determination of vectorial transport. The uptake of transferrin-bound iron (Fe) via transferrin receptor was used to determine the polarization of mammary epithelial cells. Wild-type HC11 cells were cultured on cell culture inserts in growth medium until confluent. Transepithelial resistance (TEER) was used to monitor tight junction formation, and experiments were conducted 4 days post-TEER stabilization. 59Fe-transferrin was diluted in serum-free growth medium (20 µM; Sigma) added to either the top or the bottom chamber, and then incubation was begun at 37°C for 4 h. The cells were extensively washed with ice-cold PBS, and the amount of radioactivity in the cell fraction was quantified in a gamma scintillation counter. The percentage of 59Fe uptake was calculated as (cpm in cell fraction/total cpm added to the insert) x 100, and the chamber from which the highest 59Fe-transferrin uptake was observed was denoted as the serosal membrane.
65Zn transport in HC11 cells. To determine the effects of cell differentiation on mammary epithelial cell Zn2+ transport, wild-type HC11 cells were cultured on cell culture inserts in growth medium until confluent and then cultured in differentiation medium for 2 days. TEER was used to monitor tight junction formation, and experiments were conducted 4 days post-TEER stabilization. Zn2+ transport across a monolayer of HC11 cells was assessed after addition of nondifferentiation medium (growth medium minus serum and EGF, pH 7.0) or differentiation medium, pH 7.0, containing 1 µM ZnCl2 and 0.1 µCi 65ZnCl2 (2.94 mCi/mg; Los Alamos National Laboratory, Los Alamos, NM) to the top of the cell culture insert. Cells were incubated at 37°C for 4 h. Cellular 65Zn import was determined by quantifying radioactivity in the cell fraction in a gamma scintillation counter after extensively washing the cell culture insert in ice-cold PBS containing 1 mM EDTA. 65Zn export was determined by quantifying radioactivity in the bottom chamber.
65Zn import in HC11 cells. To determine the effects of cell differentiation on Zn2+ import, confluent wild-type HC11 cells were cultured on polycarbonate plates in growth medium or treated with differentiation medium for 2 days. Medium was aspirated and replaced with nondifferentiation or differentiation medium, pH 7.0, containing 1 µM ZnCl2 and 0.1 µCi 65ZnCl2. Cells were incubated at 37°C for up to 24 h. Transfected HC11 cells were cultured on polycarbonate plates for 16 h. Transfection medium was aspirated and replaced with nondifferentiation or differentiation medium containing 1 µM ZnCl2 and 0.1 µCi 65ZnCl2. Cells were incubated at 37°C for 4 h. After being washed extensively in ice-cold PBS containing 1 mM EDTA, cells were solubilized with 1% SDS and Zn2+ uptake was determined by quantifying 65Zn in the cell fraction using a gamma scintillation counter.
Measurement of cellular Zn2+ concentration. Cultured HC11 cells were briefly rinsed with PBS, drained, and scraped into a microfuge tube. Cells were digested with 1 ml of 16 N ultrapure trace mineral-free nitric acid for 1 wk at room temperature, and Zn2+ concentration was analyzed using atomic absorption spectrophotometry as described previously (2).
Cell viability. HC11 cells were cultured in 12-well polycarbonate plates for 148 h posttransfection. Cells were detached with Trypsin-EDTA cell dissociation solution (Sigma) for 5 min at 37°C, centrifuged at 1,000 g for 5 min, and resuspended in growth medium. Viable cell number was determined using hemocytometric cell counting after Trypan blue exclusion.
Statistical analysis. Results are presented as means ± SD. Statistical comparisons were performed using Student's t-test (Prism Graph Pad, Berkeley, CA), and significance was demonstrated at P < 0.05.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
Zip3 knockdown reduced 65Zn uptake into HC11 cells. After transfection of Zip3 siRNA, Zip3 mRNA expression was reduced by 80% after 16 h, and we were unable to detect Zip3 protein in HC11 cells transfected with Zip3 siRNA (data not shown). To determine the functional consequence of reduced Zip3 expression on 65Zn uptake into mammary epithelial cells, 65Zn uptake was determined in secreting and proliferating HC11 cells. Data indicate that reduced Zip3 expression results in significantly lower 65Zn uptake (69% decrease) in secreting HC11 cells; however, although significant, this difference is not as notable in proliferating cells (42% decrease) (Fig. 5), suggesting that Zip3 contributes to enhanced Zn2+ transport in mammary epithelial cells with a secretory phenotype. Furthermore, we observed that Zip3 knockdown reduced mammary cell viability by 75% after 24 h, suggesting that uptake of Zn2+ via Zip3 is essential for mammary epithelial cell survival.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Differentiation of proliferating mammary epithelial cells to a fully functional, secretory cell type is hormonally regulated and essential for preparing these cells for secretion (3). Furthermore, once differentiated, secreting mammary epithelial cells require episodic hormonal stimulation to maintain the expression, production, and secretion of many milk components (22), similar to the requirements for galactopoeisis (25, 26). The higher Zip3 expression and 65Zn uptake in secreting mammary epithelial cells suggests enhanced Zip3-mediated Zn2+ import in actively secreting mammary epithelial cells. Furthermore, once differentiated, mammary epithelial cells exhibited a transient relocalization of Zip3 to the plasma membrane that paralleled increased 65Zn import in response to prolactin. This is reminiscent of dynamic alterations in intracellular GLUT1 compartmentalization in mammary epithelial cells in response to prolactin stimulation (12) and demonstrates the requirement for episodic Zn2+ uptake into these cells to provide Zn2+ for secretion.
Previous studies have shown that while Zip3 expression is not highly regulated by Zn2+ exposure at the transcript level (6), posttranslational regulation dominates as Zn2+ exposure rapidly distributes Zip3 to intracellular organelles while it increases in cell surface abundance in cells made Zn2+ deficient (16, 28). While a statistically significant effect of differentiation on cellular Zn2+ concentration could not be documented, cellular Zn2+ concentration was higher in secreting cells, which implies that Zip3 localization may be mediated by fluctuations in cellular Zn2+ partitioning. Whether alterations in Zip3 localization reflect direct posttranslational modifications of Zip3, secondary effects on intracellular signaling, or altered cellular organization is not yet known; nevertheless, the predicted amino acid sequence of Zip3 indicates several possible ubiquitination sites, which suggests posttranslational regulation of Zip3 and may include proteosomal degradation similar to that exhibited by the yeast Zn2+ importer ZRT1 (11), particularly because cellular differentiation of HC11 cells increases expression of the proteasome machinery (3).
In summary, our data indicate that Zip3 plays a major role in mammary epithelial cell Zn2+ uptake and that actively secreting mammary epithelial cells have an enhanced dependence on Zip3-mediated Zn2+ import, providing a physiological explanation for the restricted tissue distribution of this Zn2+ importer. Finally, prolactin stimulation of Zip3 trafficking between the plasma membrane and an intracellular compartment may help to link lactogenic hormone secretion with episodic mammary gland nutrient uptake and transient milk nutrient secretion to provide an adequate amount of Zn2+ for the suckling neonate.
![]() |
FOOTNOTES |
---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. Clegg MS, Keen CL, Lönnerdal B, and Hurley LS. Influence of ashing techniques on the concentration of trace elements in animals tissues: 1. Wet ashing. Biol Trace Elem Res 3: 107115, 1981.[ISI]
3. Desrivières S, Prinz T, Castro-Palomino Laria N, Meyer M, Boehm G, Bauer U, Schäfer J, Neumann T, Shemanko C, and Groner B. Comparative proteomic analysis of proliferating and functionally differentiated mammary epithelial cells. Mol Cell Proteomics 2: 10391054, 2003.
4. Doppler W, Villunger A, Jennewein P, Brduscha K, Groner B, and Ball RK. Lactogenic hormone and cell type-specific control of whey acidic protein gene promoter in transfected mouse cells. Mol Endocrinol 5: 16241632, 1991.[Abstract]
5. Dufner-Beattie J, Kuo YM, Gitschier J, and Andrews GK. The adaptive response to dietary zinc in mice involves the differential cellular localization and zinc regulation of the zinc transporters ZIP4 and ZIP5. J Biol Chem 279: 4908249090, 2004.
6. Dufner-Beattie J, Langmade SJ, Wang F, Eide D, and Andrews GK. Structure, function, and regulation of a subfamily of mouse zinc transporter genes. J Biol Chem 278: 5014250150, 2003.
7. Eide DJ. The SLC39 family of metal ion transporters. Pflügers Arch 447: 796800, 2004.[CrossRef][ISI][Medline]
8. Gaither LA and Eide DJ. Functional expression of the human hZIP2 zinc transporter. J Biol Chem 275: 55605564, 2000.
9. Gaither LA and Eide DJ. The human ZIP1 transporter mediates zinc uptake in human K562 erythroleukemia cells. J Biol Chem 276: 2225822264, 2001.
10. Geley S and Müller C. RNAi: ancient mechanism with a promising future. Exp Gerontol 39: 985998, 2004.[CrossRef][ISI][Medline]
11. Gitan RS and Eide DJ. Zinc-regulated ubiquitin conjugation signals endocytosis in the yeast ZRT1 transporter. Biochem J 346: 329336, 2000.[CrossRef][ISI][Medline]
12. Haney PM. Localization of the GLUT1 glucose transporter to brefeldin A-sensitive vesicles of differentiated CIT3 mouse mammary epithelial cells. Cell Biol Int 25: 277288, 2001.[ISI][Medline]
13. Huang L and Gitschier J. A novel gene involved in zinc transport is deficient in the lethal milk mouse. Nat Genet 17: 292297, 1997.[ISI][Medline]
14. Huyer G, Piluek WF, Fansler Z, Kreft FG, Hochstrasser M, Brodsky JL, and Michaelis S. Distinct machinery is required in Saccharomyces cerevisiae for the endoplasmic reticulum-associated degradation of a multispanning membrane protein and a soluble luminal protein. J Biol Chem 279: 3836938378, 2004.
15. Kambe T, Narita H, Yamaguchi-Iwai Y, Hirose J, Amano T, Sugiura N, Sasaki R, Mori K, Iwanaga T, and Nagao M. Cloning and characterization of a novel mammalian zinc transporter, ZnT-5, abundantly expressed in pancreatic -cells. J Biol Chem 277: 1904955, 2002.
16. Kelleher SL and Lönnerdal B. Zn transporter levels and localization change throughout lactation in rat mammary gland and are regulated by Zn in mammary cells. J Nutr 133: 33783385, 2003.
17. Kelleher SL and Lönnerdal B. Zinc transporters in the mammary gland respond to marginal zinc and vitamin A intake during lactation in rats. J Nutr 132: 32803285, 2002.
18. King JC. Enhanced zinc utilization during lactation may reduce maternal and infant zinc depletion. Am J Clin Nutr 75: 23, 2002.
19. McMahon RJ and Cousins RJ. Regulation of the zinc transporter ZnT-1 by dietary zinc. Proc Natl Acad Sci USA 95: 48414186, 1998.
20. Murgia C, Vespignani I, Cerase J, Nobili F, and Perozzi G. Cloning, expression, and vesicular localization of transporter Dri 27/ZnT4 in intestinal tissue and cells. Am J Physiol Gastrointest Liver Physiol 277: G1231G1239, 1999.
21. Neville MC. Physiology of lactation. Clin Perinatol 26: 251279, 1999.[ISI][Medline]
22. Neville MC, McFadden TB, and Forsyth I. Hormonal regulation of mammary differentiation and milk secretion. J Mammary Gland Biol Neoplasia 7: 4966, 2002.[CrossRef][ISI][Medline]
23. Palmiter RD, Cole TB, and Findley SD. ZnT-2, a mammalian protein that confers resistance to zinc by facilitating vesicular sequestration. EMBO J 15: 17841791, 1996.[Abstract]
24. Palmiter RD and Huang L. Efflux and compartmentalization of zinc by members of the SLC30 family of solute carriers. Pflügers Arch 447: 744751, 2004.[CrossRef][ISI][Medline]
25. Rillema JA and Hill MA. Prolactin regulation of the pendrin-iodide transporter in the mammary gland. Am J Physiol Endocrinol Metab 284: E25E28, 2003.
26. Rillema JA, Houston TL, and John-Pierre-Louis K. Prolactin, cortisol, and insulin regulation of nucleoside uptake into mouse mammary gland explants. Exp Biol Med (Maywood) 228: 795799, 2003.
27. Shennan DB and Peaker M. Transport of milk constituents by the mammary gland. Physiol Rev 80: 925951, 2000.
28. Wang F, Dufner-Beattie J, Kim BE, Petris MJ, Andrews GK, and Eide DJ. Zinc-stimulated endocytosis controls activity of the mouse ZIP1 and ZIP3 zinc uptake transporters. J Biol Chem 279: 2463124639, 2004.
29. Wang F, Kim BE, Petris MJ, and Eide DJ. The mammalian ZIP5 protein is a zinc transporter that localizes to the basolateral surface of polarized cells. J Biol Chem 279: 5143351441, 2004.
|
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Visit Other APS Journals Online |