During lactation, a substantial amount of Zn2+ is transferred by the mammary gland from the maternal circulation into milk; thus secretory mammary epithelial cells must tightly regulate Zn2+ transport to ensure optimal Zn2+ transfer to the suckling neonate. To date, six Zn2+ import proteins (Zip1–6) have been identified; however, Zip3 expression is restricted to tissues with unique requirements for Zn2+, such as the mammary gland, which suggests that it may play a specialized role in this tissue. In the present study, we have used a unique mammary epithelial cell model (HC11) to characterize the role of Zip3 in mammary epithelial cell Zn2+ transport. Confocal microscopy demonstrated that Zip3 is localized to the cell surface in mammary epithelial cells and transiently relocalized to an intracellular compartment in cells with a secretory phenotype. Total 65Zn transport was higher in secreting cells, while gene silencing of Zip3 decreased 65Zn uptake into mammary epithelial cells, particularly in those with a secretory phenotype. Finally, reduced expression of Zip3 ultimately resulted in cell death, indicating that mammary epithelial cells have a unique requirement for Zip3-mediated Zn2+ import, which may reflect the unique requirement for Zn2+ of this highly specialized cell type and thus provides a physiological explanation for the restricted tissue distribution of this Zn2+ importer.
- zinc transport
- mammary gland
zinc is the second most abundant trace element in the human body (13). As an essential mineral, Zn2+ is required for many proteins involved in DNA and protein synthesis, mitosis, and cell division, serving both a structural and a catalytic role. Participation in such diverse cellular processes highlights the need to tightly regulate Zn2+ homeostasis; thus the activity of Zn2+-specific transport proteins that control cellular Zn2+ uptake and efflux is central to this regulation. Recently, a number of proteins have been described that participate in Zn2+ trafficking across membranes, and they are divided into two distinct families. In mammals, members of the ZnT family (ZnT1–7) function to transport Zn2+ from the cytosol, either across the plasma membrane or into vesicles (24). The second family of mammalian Zn2+ transporters (Zip1–6) has been identified as a result of gene sequence homology with known Zn2+ transporters (ZRT1, IRT1-like protein) found in plants and yeast (8), and these have been shown to facilitate cellular Zn2+ uptake in transfected cell models (6, 29). Although Zip1 expression is ubiquitous, abundant expression of Zip2–6 is tissue-specific (5, 28, 29), suggesting that these proteins play tissue-specific roles in cellular Zn2+ metabolism.
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 (0.5–1 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
Cell culture and transient transfection.
Mouse mammary epithelial cells (HC11) were a gift from Dr. Jeffrey M. Rosen (Dept. of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX) and used with the permission of Dr. Bernd Groner (Institute for Biomedical Research, Frankfurt, Germany). HC11 cells were seeded into polycarbonate dishes and cultured in growth medium (RPMI 1640; Life Technologies, Rockville, MD) supplemented with 10% fetal bovine serum (Sigma, St. Louis, MO), gentamicin (50 mg/l; Sigma), insulin (5 mg/l; Sigma), and epidermal growth factor (EGF, 10 μg/l; Sigma) at 37°C in 5% CO2. To differentiate HC11 cells into cells with a secretory phenotype, HC11 cells were cultured in differentiation medium (growth medium minus serum and EGF containing 1 μg/ml prolactin and 1 μM cortisol) for up to 48 h. For transient transfection, cells were seeded onto 12-well polycarbonate plates at a density of 5 × 105 cells/well and transfected 24 h later with 1.6 μg of Zip3 small interfering RNA (siRNA) (sense, 5′-GGUCAUCGAGGCUGACUUGTT-3′; antisense, 5′-CAAGUCAGCCCUCGAUGACCTT-3′) (Ambion, Austin, TX) or a control scrambled siRNA (Ambion) in transfection medium (growth medium minus serum and gentamicin) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions.
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 ×100 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 ΔCt = 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(ΔΔCt), where ΔΔCtGENE = mean ΔCtZip3 control − ΔCtZip3 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) × 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).
HC11 cells were cultured in 12-well polycarbonate plates for 1–48 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.
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.
Zip3 is localized to the plasma membrane and a perinuclear compartment in HC11 cells.
Zip3 has been functionally characterized as a Zn2+ import protein in transfected cells (6). Using confocal microscopy, we determined that Zip3 colocalized extensively with DiI (Fig. 1, A–C), indicating that Zip3 is localized primarily at the cell surface in proliferating HC11 cells (Fig. 2A). Conversely, in secreting cells, Zip3 intensely stained a perinuclear compartment (Fig. 2B), and we observed little overlap with DiI (Fig. 1, D–F) and minimal overlap with transferrin (Fig. 1, J–L), showing that Zip3 translocates primarily to a nonendosomal intracellular compartment in differentiated, secreting mammary cells. Furthermore, once cells were differentiated, we observed a transient increase in Zip3 abundance at the cell surface (Fig. 2, C–E) in response to additional stimulation with prolactin (Fig. 1, G–I). These results demonstrate an effect of differentiation to a secretory phenotype, as well as an effect of episodic stimulation, on Zip3 localization.
Zinc uptake is transiently increased in secreting HC11 cells.
Cell polarization studies allowed us to determine that the top chamber represented the serosal cell surface in HC11 cells grown in bicameral chambers, because 91 ± 2% of 59Fe-transferrin was taken up across this membrane while only 0.3 ± 0.1% of 59Fe-transferrin was taken up across the bottom chamber, and we observed no effect of differentiation on this process (data not shown). These results document the direction of vectorial transport that occurs in these polarized cells. We then compared Zn2+ uptake in proliferating and secreting cells and observed that Zn2+ uptake in proliferating HC11 cells gradually increased over 16 h and then remained relatively constant, while Zn2+ uptake in secreting cells was transiently higher, peaking after 8 h and then declining to basal levels (Fig. 3). Zn2+ export into the bottom chamber after 8 h was significantly higher (P < 0.05) in secreting cells (68.5 ± 4.6% of total 65Zn added) than in proliferating cells (48.7 ± 6.3% of total 65Zn added), indicating that differentiation positively affects Zn2+ transport activity and that mammary epithelial cells with a secretory phenotype have transiently enhanced Zn2+ uptake.
Zip3 expression is higher in differentiated HC11 cells.
To determine whether enhanced Zn2+ import in secreting mammary epithelial cells was associated with higher Zip3 expression, relative Zip3 mRNA and protein levels were determined using real-time PCR and Western blot analysis, respectively. Our data demonstrate that Zip3 mRNA (P < 0.05) and protein levels (Fig. 4) in secreting cells were twofold those of proliferating cells. Interestingly, the increase in Zip3 protein appears to be transient, indicating that while secreting mammary epithelial cells have higher overall Zip3 expression than do proliferating mammary cells, further stimulation with prolactin transiently decreased Zip3 protein levels, suggesting additional posttranscriptional regulation. Cellular Zn2+ concentration in secreting cells (1.1 ± 0.08 nmol/106 cells) was not significantly different (P = 0.14) from that in proliferating cells (0.87 ± 0.02 nmol/106 cells).
Cycloheximide treatment reduced Zip3 protein levels but increased Zip3 mRNA levels.
To determine whether Zip3 gene expression required active Zip3 protein translation, HC11 cells were treated with cycloheximide for 48 h and Zip3 mRNA and protein levels were determined using real-time PCR and Western blot analysis. We determined that while Zip3 protein levels were reduced 86% by cycloheximide treatment, Zip3 mRNA levels were 1.8-fold higher, suggesting that protein translation is not required for Zip3 gene expression.
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.
Within the mammary gland, the highly specialized secretory mammary epithelial cell is responsible for the secretion of milk components in a vectorial manner and thus facilitates the transport of large amounts of Zn2+ from maternal circulation into milk to provide the neonate with an optimal amount of dietary Zn2+. Zip3 has been reported to be expressed in many tissues (28), and a cursory examination of the expressed sequence tag (EST) database suggests that it is abundantly expressed. However, similar to the results of Northern blot analysis of mouse tissues (6), further analysis indicates that ESTs appear to be restricted to highly specialized tissues that have an unusually high and unique requirement for Zn2+ (i.e., mammary gland, brain, eye, pancreas, thymus), which leads us to speculate that Zip3 may perform a special role in these tissues. Similar to Zip3 localization in lactating rat mammary gland (16), the results of the present study document the localization of Zip3 to the plasma membrane of mammary epithelial cells, indicating that Zip3 may facilitate mammary epithelial cell Zn2+ import (6). The use of gene silencing techniques has greatly aided our understanding of many complex biological processes (10) and is becoming an increasingly common tool in evaluating protein functionality and essentiality in specific cell types. The decreased Zn2+ uptake observed as a result of Zip3 knockdown demonstrates that Zip3 facilitates Zn2+ import in mammary epithelial cells. Furthermore, decreased cell viability demonstrates the biological essentiality of Zip3 in mammary epithelial cells and may reflect the unique requirement for enhanced Zn2+ transport via Zip3 in this highly specialized cell type. However, the facts that cell death did not occur concomitantly with significant reductions in Zip3 expression and that residual Zn2+ uptake was detected may reflect the observation that Zip1 is also expressed in mammary epithelial cells (data not shown). Thus our results may document a two-tiered system of Zn2+ uptake in cells in which both proteins are expressed as proposed by Wang et al. (28). We speculate that Zip1 expression may be adequate to maintain cellular Zn2+ levels, particularly in proliferating cells, because Zip3 knockdown in proliferating mammary epithelial cells did not elicit as dramatic an effect on Zn2+ uptake (only ∼42% reduction), while Zip3 knockdown in secreting cells reduced Zn2+ uptake by 69%, suggesting an enhanced reliance on Zip3 for Zn2+ uptake in secreting mammary epithelial cells. However, as cellular Zn2+ requirements may be enhanced in secreting cells or as cellular Zn2+ level becomes compromised, cell death rapidly ensues in the absence of adequate Zip3 expression.
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.
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