![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Department of Nutrition, University of California Davis, Davis, CA 95616
1To whom correspondence should be addressed. E-mail: slkelleher{at}ucdavis.edu.
 |
ABSTRACT |
|---|
 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
KEY WORDS: • mammary gland • zinc • lactation • zinc transporter
Zinc (Zn) is a nutrient required for many proteins involved in DNA synthesis, protein synthesis, mitosis and cell division. During lactation in humans, a substantial amount of Zn is transported across the mammary gland into milk (0.5–1 mg/d), almost twice the amount that is transferred daily across the placenta to the fetus during pregnancy (1), indicating that mammary gland Zn transport is a very active process. Furthermore, milk Zn concentration is maintained over a wide range of dietary Zn intake (2,3), which suggests that mammary gland Zn import and export are tightly coordinated to provide adequate Zn to the suckling neonate. Interestingly, although plasma Zn concentration increases, milk Zn concentration decreases throughout the normal course of lactation in both rodents and humans (4); however, the transport mechanisms that regulate this longitudinal decrease are not well understood (5).
Recently, a number of mammalian proteins that participate in Zn trafficking across membranes were described (6); these are divided into two distinct families. The ZnT family of Zn transporters is a member of the larger cation diffusion facilitator family and currently contains seven members (ZnT-1 through ZnT-7). With the exception of ZnT-5 (7), they are structurally similar having six transmembrane domains and a histidine-rich domain that is believed to play a key role in Zn binding; however, the specific mechanisms these transporters utilize to transport Zn are not known. We described previously the expression of ZnT-1, ZnT-2 and ZnT-4 in the mammary gland of lactating rats and have documented changes in mRNA and protein levels in response to marginal Zn deficiency that we believe participate in the maintenance of milk Zn levels despite inadequate Zn intake (8). ZnT-1 is localized primarily at the plasma membrane, is expressed ubiquitously and functions to export Zn from the cytosol (9). Both ZnT-2 and ZnT-4 are associated with the plasma membrane and may export Zn from the cytosol into intracellular vesicles (10,11); however, the physiologic significance of vesicle Zn sequestration remains obscure.
The second family of mammalian Zn transporters (Zip1–4) was identified as a result of gene sequence homology with known Zn transporters (ZRT1, IRTl-like protein) found in plants and yeast (12) that have been shown to facilitate cellular Zn uptake. Although the expression of ZIP1 is ubiquitous (13), ZIP2 expression appears to be restricted (12,14). Cell transfection studies determined that Zip1 and Zip2 are localized to both the plasma membrane (K562 cells) and intracellular vesicles (COS-7 cells), suggesting that they may have cell-specific roles in Zn import (13,15). ZIP4 expression is restricted to the visceral yolk sack and the intestinal tract in mice where it is inversely related to Zn status (16). Furthermore, several genetic mutations in ZIP4 were identified recently in patients with acrodermatitis enteropathica, a genetic disorder of severe Zn deficiency (17). Zip3 was identified in mouse mammary gland (Genebank Accession # NM134135), suggesting that it plays a role in mammary gland Zn import. However, to our knowledge, the physiologic regulation of many of the Zip proteins has not yet been described.
In this study, we used the lactating rat as a model and characterized changes in ZnT-1, ZnT-2, ZnT-4 and Zip3 mRNA and protein levels that occur in the mammary gland throughout lactation to elucidate potential mechanisms that may be responsible for the longitudinal decrease in milk Zn concentration that has been observed in both rodents and humans. Furthermore, because we determined that mammary gland Zn concentration increases throughout lactation, we used cultured mouse mammary epithelial cells as a model and determined effects of increased cellular Zn levels on mediating these changes.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
This study was approved by Animal Research Services at the University of California, Davis, which is accredited by the American Association for the Accreditation of Laboratory Animal Care. Virgin Sprague-Dawley rats (n = 50; 
250 g) were obtained commercially (Simonsen, Gilroy, CA); the rats consumed an AIN-93 casein-based semipurified experimental diet ad libitum. They were maintained in stainless steel hanging cages on a 12-h light:dark cycle for 3 wk and bred. Upon pregnancy confirmation, rats were moved to plastic cages containing shaved wood bedding. On postnatal d 1, litters were culled to 8 pups. On lactation day (LD) 1, 5, 10, 15 and 20, 6 dams/d were removed from pups for 4 h, anesthetized (intraperitoneally, 1.6 mg xylazine/kg and 33 mg ketamine/kg), and a complete milk collection (3 mL) was obtained manually from inguinal mammary glands within 10 min after oxytocin injection (subcutaneous, 10 U/dam). Whole blood was removed via cardiac puncture and collected into heparinized vials and rats were killed by asphyxiation with CO2. Plasma was separated by centrifugation at 2000 x g for 15 min at 4°C and frozen at –80°C until analyzed for Zn concentration. Mammary glands were dissected and immediately snap-frozen in liquid nitrogen for determination of Zn concentration and Zn transporter protein levels or homogenized in TriZOL (Gibco Life Technologies, Rockville, MD) for RNA isolation. An additional 4 rats/d were removed from their pups for 4 h, killed by asphyxiation with CO2 and mammary glands were dissected and fixed in paraformaldehyde (40 g/L) in PBS, pH 7.4 for immunostaining as previously described (8).
Cell culture.
Mouse mammary epithelial cells (HC11) were a gift from Dr. Jeffrey Rosen (Houston, Texas) and used with permission of Dr. Bernd Groner (Institute for Biomedical Research, Frankfurt, Germany). HC11 cells are a clonal derivative of the COMMA-1D cell line; they are uniquely responsive to lactogenic hormones and have been proven to be an important model for understanding hormonal regulation of mammary cell differentiation and milk protein gene expression and protein secretion (18). HC11 cells were seeded onto polycarbonate dishes (RNA isolation) or cell culture inserts (Zn transport) and grown to postconfluence in Growth Medium [RPMI 1640 medium (Gibco Life Technologies) supplemented with fetal bovine serum (100 g/L, Sigma, St. Louis, MO), gentamycin (Sigma), insulin (Sigma) and epidermal growth factor (Sigma)] at 37°C and 5% CO2.
Zinc treatment of HC11 cells.
Postconfluent cells were washed with PBS and incubated in Induction Medium [RPMI 1640 medium containing gentamycin and insulin] with or without supplemental Zn (15 µmol/L as ZnSO4) for 16 h at 37°C and 5% CO2. After Zn treatment, cells were solubilized in TriZOL (Gibco Life Technologies) for RNA isolation or used for Zn transport studies.
Zn transport in HC11 cells.
Transepithelial resistance (TEER) was used to monitor tight junction formation across a monolayer of HC11 cells grown on cell inserts, and experiments were conducted 4 d post-TEER stabilization. Cellular Zn transport was assessed after the addition of Induction Medium containing 0.1 µCi (35 KBq) 65Zn (Los Alamos National Laboratory, Los Alamos, NM) to the top of the cell culture insert. Zn uptake into the cell and Zn export into the bottom chamber were determined by quantifying radioactivity in the cell fraction and bottom chamber in a gamma scintillation counter after 8 h.
Immunofluorescence of HC11 cells.
HC11 cells were seeded onto glass coverslips and cultured for 2 d in Growth Medium. Cells were washed in PBS and incubated in Induction Medium containing Zn (0 or 15 µmol/L ZnSO4) for 16 h at 37°C. Medium was aspirated and cells were washed extensively with PBS, fixed in paraformaldehyde (40 g/L)/PBS, pH7.4 for 30 min, washed in PBS, then permeabilized with Triton X-100 (4 g/L)/PBS for 4 min. Nonspecific binding was blocked in PBS/goat serum (100 g/L)/bovine serum albumin (10 g/L) for 60 min followed by incubation with primary antiserum (Zip3, ZnT-1, ZnT-2 or ZnT-4; 1:100) for 45 min with rocking at room temperature. After extensive washing with PBS/Tween-20 (0.5 g/L), primary antibody was detected using Alexa 488-conjugated goat and rabbit IgG (1 mg/L, Molecular Probes, Eugene, OR) for 45 min at room temperature with rocking, shielded from light. Stained cells were washed extensively in PBS/Tween-20; coverslips were drained and mounted in glycerol/PBS and sealed with nail polish. Co-localization of Zn transporters to the endosomal compartment was studied after incubation of HC11 cells with Alexa 546-conjugated transferrin (Molecular Probes) at 37°C for 10 min. Immunofluorescent imaging was performed using an Olympus BX50WI, with UPlanApo 100X oil lens N.A. 1.35, and digital images were captured using the BioRad Radiance 2100 confocal system, utilizing LaserSharp2000, version 4.1 (BioRad, Hercules, CA).
Zinc analysis.
Zinc concentrations of plasma, milk and mammary gland were measured as previously described (8). Cultured cells were scraped and transferred to a microfuge tube and digested with 1 mL of 16 mol/L ultrapure trace mineral–free nitric acid for 1 wk at room temperature.
cDNA probes synthesis and RNA analysis by Northern blotting.
cDNA probes to ZnT-1, ZnT-2 and ZnT-4 were produced by PCR and labeled with 32P as previously described (8). Using data mining with the human ZIP1 sequence (Genebank Accession #NM 014437), we identified a homologous murine sequence (ZRT1, IRTl-like protein 3, Genebank Accession # NM134135) expressed in mouse mammary gland, which was further used to search the rat expressed sequence tag database. A clone (MGC:7461) was identified and purchased from American Type Culture Collection (Manassus, VA), amplified, isolated and purified as previously described (8).
Equal amounts of mRNA from individual mammary glands (4 µg), isolated from total RNA using the Microfast Track Kit (Invitrogen, Carlsbad, CA), or total RNA (20 µg) isolated from cultured cells were electrophoresed and hybridized as previously described (8). Northern blots were probed with glyceraldehyde-3-phosphate dehydrogenase cDNA for normalization.
Preparation of mammary gland protein extracts and membrane fractions.
Mammary gland total protein was extracted as described previously (8). The postnuclear supernatant was isolated by centrifugation for 5 min at 500 x g at 4°C and centrifuged at 15,000 x g for 10 min at 4°C (pellet 1), followed by 100,000 x g for 45 min at 4°C (pellet 2) then 100,000 x g for 4 h at 4°C (pellet 3) to isolate crude fractions of large membrane vesicles, microsomes and small vesicles, respectively (19). The membrane pellets were resuspended in homogenization buffer and protein concentration was determined by the Bradford protein assay (BioRad). N-linked protein glycosylation of both high-mannose and complex oligosaccharides was determined after incubation with EndoH and PNGase, respectively. Mammary gland protein (100 µg) was denatured in Laemmli sample buffer containing ß-mercaptoethanol (10 g/L) and boiled for 10 min. The sample was diluted 1:10 with 50 mmol/L sodium acetate, pH 6.5, and 15 mU EndoH (Sigma) or 1:10 with buffer (50 mmol/L Tris, pH 7.5, 150 mmol/L NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS) and 2.5 U PNGase (New England Biolabs, Beverly, MA). Samples were incubated at 37°C for 1 h and resolved by SDS-PAGE.
Production and verification of antiserum.
A peptide fragment of Zip3 (FRRERPPFIDLETFNAGSDAGSDSEYESPF) was synthesized (Genemed Synthesis, South San Francisco, CA) with an additional cysteine residue for conjugation to keyhole limpet hemocyanin at the C-terminal end. The sequence was verified by amino acid analysis and MS. Antisera to ZnT-1, ZnT-2 and ZnT-4 were produced as previously described (8). Specificity of peptide antisera was verified by the disappearance of unique immunoreactive bands after co-incubation with peptide (1 mg) and the appearance of specific bands not detected on a Western blot of mammary gland protein after incubation with preimmune serum (see below).
Immunoblotting.
Equal amounts of mammary gland protein (100 µg) were diluted 1:1 in Laemmli sample buffer with or without 1% ß-mercaptoethanol and resolved by SDS-PAGE (12%). Mammary gland protein was transferred to nitrocellulose for 90 min at 350 mA. Blots were blocked overnight at 4°C with 5% nonfat milk in PBS/0.1% Tween-20 (PBS-T) and washed 3 times in PBS-T. Blots were detected and visualized as described previously (8) and quantified using the Chemi-doc Gel Quantification System (BioRad). Blots were stripped and reprobed for ß-actin as a loading control.
Immunostaining of mammary gland.
Fixed mammary glands from rats at LD 1, 5, 10, 15 and 20 were immunostained as described previously (8).
Statistical analysis.
The results of animal experiments are presented as means ± SD, n = 6 dams/d. Statistical comparisons of animal experiments were made using one-way ANOVA and post-tested using the Tukey-Kramer test (Prism Graph Pad, Berkeley, CA). Results of cell culture experiments are presented as means ± SD. Statistical comparisons of cultured cell experiments were made using Student’s t test. Data presented represent three independent experiments with treatments and analyses done in triplicate. Significance was demonstrated at P < 0.05.
|
RESULTS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
Plasma and mammary gland Zn concentration increased, whereas milk Zn concentration gradually decreased throughout the course of lactation P < 0.05 (Table 1), indicating that the mammary gland stringently regulates the amount of Zn secreted into milk and suggesting that the decrease in milk Zn level is not a consequence of mammary gland Zn depletion.
|
The antisera specificity was verified by the appearance of unique protein bands not observed in immunoblots of mammary gland protein incubated with preimmune serum and by the elimination of bands after co-incubation of the rabbit antiserum with peptide antigen (Fig. 1).
|
In addition to ZnT-1, ZnT-2 and ZnT-4, ZIP3 was expressed in the mammary gland and its gene product was localized to the basolateral membrane of mammary epithelial cells, thus potentially participating in Zn import into the mammary gland from the maternal circulation. Additionally, changes in mRNA and protein levels of these Zn transporters that may play a functional role in decreasing milk Zn levels as lactation progresses occurred throughout the course of lactation. Mammary gland mRNA levels of ZnT-1, ZnT-2 and ZIP3 were significantly altered to different extents as lactation progressed (Fig. 2A and B). Although both ZnT-1 and ZnT-2 mRNA levels increased through LD10, ZnT-1 levels remained elevated, whereas ZnT-2 mRNA levels decreased through LD20. ZnT-4 mRNA level remained relatively constant and the ZIP3 mRNA level decreased through LD15. The fact that these changes in gene expression cannot directly explain the reduction in milk Zn levels throughout lactation suggests that post-transcriptional regulation must play a role in this process.
|
60 and 50 kDa (Fig. 3), both of which increased throughout lactation (Table 2), P < 0.05. Two minor ZnT-1 proteins at 40 and 34 kDa were also detected but were unaffected by the length of lactation (data not shown). Incubation of mammary gland extract with EndoH or PNGase did not result in a change in molecular weight of any ZnT-1 protein (data not shown). However, under nonreducing conditions, the 50- and 60-kDa proteins participated in the formation of two ZnT-1 complexes at 158 and 71 kDa, and the levels of both ZnT-1 complexes changed longitudinally, P < 0.05 (Fig. 4A). Using differential centrifugation we further determined that the ZnT-1 complexes were localized to different subcellular compartments (Fig. 5), although some overlap was detected, and this localization did not change over the course of lactation (data not shown).
|
|
|
|
52 and 45 kDa, which did not form a complex under nonreducing conditions; although the larger 52-kDa protein decreased throughout lactation (Table 2), P < 0.05, the level of the smaller protein was unaffected (Fig. 3). Deglycosylation experiments with EndoH and PNGase indicated that the 52-kDa protein was a result of extensive N-linked glycosylation of the 45-kDa protein (Fig. 6).
|
The calculated molecular weight of Zip3 based on the predicted protein sequence of mouse Zip3 (Accession # NM13435) is 37 kDa, and it is predicted to have one N-linked glycan at Asn142. In the rat mammary gland, we detected one Zip3 protein at 70 kDa (Fig. 3), which decreased through LD15, then increased at LD20 (Table 2), P < 0.05, and did not form a complex under nonreducing conditions (data not shown). Deglycosylation experiments did not result in N-glycan cleavage (data not shown).
Zn transporter localization changes throughout lactation.
ZnT-4 stains in primarily a punctate pattern, suggesting a vesicular localization in mammary epithelial cells. Intense staining was associated with the apical membrane at LD1 and LD5; however, the staining pattern appeared more equally distributed between the apical and basolateral membranes from LD10 to 20 (Fig. 7A). These results suggest that ZnT-4 may participate in Zn export into milk to a greater extent during early lactation. Similar to ZnT-4, ZnT-2 stained in primarily a punctate pattern and was localized to both the apical and basolateral membrane of mammary epithelial cells. The intensity of the apical membrane staining was particularly high during early lactation and became more faint as lactation progressed, whereas the staining intensity at the basolateral membrane did not appear to change (Fig. 7B). This suggests that ZnT-2 may also participate in Zn export into milk to a greater extent during early lactation. ZnT-1 was localized to both a vesicular compartment and the plasma membrane as visualized by both punctate and discrete staining patterns (Fig. 7C). During early lactation (LD5), ZnT-1 was detected only along the apical membrane, suggesting that in early lactation, ZnT-1 participates directly in Zn export into milk. Zip3 was localized to the basolateral membrane of mammary epithelial cells, suggesting that it participates in Zn import from maternal circulation in these cells. Paralleling results from Western blots, the intensity of Zip3 staining decreased through lactation and was detected only by immunostaining through LD10 (Fig. 7D).
|
Cells treated with 15 µmol/L ZnSO4 had higher cellular Zn concentration (0.28 ± 0.05 vs. 0.18 ± 0.04 µg/g), lower 65Zn uptake into cells and higher 65Zn export into the bottom chamber of HC11 cells cultured on cell culture inserts (Table 3). Furthermore, cells treated with 15 µmol/L ZnSO4 had higher ZnT-1 and ZnT-2 mRNA levels (P < 0.05) and lower ZIP3 mRNA levels (P < 0.05), whereas ZnT-4 mRNA levels were not affected, P = 0.08 (Fig. 8). Localization of these Zn transporters in HC11 cells was also affected by Zn treatment (Fig. 9A–L). Low cellular Zn levels were associated with Zip3 localization at the plasma membrane, whereas Zn treatment altered the localization of Zip3 to an intracellular compartment. In cells exposed to low Zn concentration, perinuclear staining of ZnT-1 was detected, whereas Zn treatment dispersed this localization throughout the cell. ZnT-2 localization was most dramatically affected by Zn treatment in that it stained very homogeneously throughout the entire untreated cell, whereas Zn treatment restricted its localization to the perinuclear region. Intense ZnT-4 staining was detected within the cell and particularly in the perinuclear region in untreated cells, whereas Zn treatment dispersed this localization to an intracellular, punctate staining pattern.
|
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
50% of normal) (21) and that pup survival can be improved by maternal Zn supplementation indicates that the mammary gland can utilize other Zn transport mechanisms to facilitate milk Zn export. Under normal circumstances in humans and rodents, milk Zn concentration decreases throughout lactation, and we hypothesized that this decrease in milk Zn level is a result of changes in Zn transporter expression or localization. Although the biological implication of each of these Zn transport mechanisms on facilitating the decline in milk Zn level cannot be definitively determined without the use of tissue-specific knock-out models, results from this study documented an association between specific changes in the mRNA and protein levels and cellular localization of Zn transporters and decreasing milk Zn levels as lactation progresses. Furthermore, data from the use of mammary epithelial cells in this study indicated that Zn plays a regulatory role in this process.
As has been observed during lactation in humans (22), the low plasma Zn concentration of lactating rats increased to a prepregnancy level as lactation progressed. Concurrent with the increasing plasma Zn level, mammary gland Zn concentration, ZnT-1 and ZnT-2 mRNA increased, whereas ZnT-4 mRNA expression was somewhat refractory, similar to observations in other tissues such as the small intestine and kidney of Zn-supplemented rats (23). However, although ZnT-1 mRNA expression was positively correlated with mammary gland Zn throughout lactation, the increase in the ZnT-2 mRNA level was transient, supporting the contention that ZnT-1, ZnT-2 and ZnT-4 are regulated by disparate mechanisms (23). Thus far, a metal responsive element has been identified only in the promoter region of ZnT-1 (24); therefore, the role Zn plays in the mechanisms regulating other Zn transporters remains to be elucidated. The extrapolation of information obtained from our studies of HC11 cells in culture suggests that the changes in ZnT-1 and ZnT-2 mRNA levels throughout lactation may be a direct consequence of increased cellular Zn concentration, as was observed in other cell models (24). In contrast, as was observed for ZIP2 (14) and ZIP4 (16), ZIP3 mRNA expression decreased as mammary gland Zn concentration increased, suggesting that ZIP3 gene expression and protein levels may be down-regulated by high Zn levels to prevent cellular Zn overload. Studies are currently underway to test this hypothesis further.
The results from these studies suggest that post-transcriptional or post-translational regulation of these Zn transporters (11) may also play a role in regulating milk Zn levels throughout lactation as was reported for other Zn (25) and copper transporters (26–28). Although progress has been made in identifying novel Zn-specific transport proteins, there is surprisingly little information regarding their molecular properties or physiologic regulation. Consistent with observations of others regarding ZnT-4 in cultured human mammary cells (29) and transfected COS-7 cells (11), we observed multiple ZnT-4 proteins similar in size to those reported by others, and further confirmed that they did not appear to be products of simple N-linked glycosylation (11). A unique feature of ZnT-4 is a leucine zipper motif in the N-terminal region of the protein [Leu68(X)6 Leu75(X)6 Leu82(X)6 Leu89], which has been postulated to participate in protein-protein interactions (11) and thus may play a role in mediating ZnT-4 protein interactions. We determined that ZnT-4 formed a 142-kDa complex whose level decreased throughout the course of lactation in the absence of ß-mercaptoethanol. The high level of the ZnT-4 protein complex during early lactation and its subsequent decline as lactation continues provides a mechanistic explanation behind the decreasing milk Zn levels that were observed. Similar to observations that were made for intestinal ZnT-4 (11), ZnT-4 in the mammary gland was localized to both basolateral and apical compartments of mammary cells; however, its relative distribution between these compartments changed throughout lactation. During early lactation, intense staining was observed in association with the apical membrane, again suggesting that it plays an important role in exporting Zn into milk during early lactation. However, during late lactation, ZnT-4 localization was more evenly distributed to both sides of the cell as well as to an intracellular vesicular compartment, possibly reducing its overall contribution to milk Zn export. Similar differences in the localization of ZnT-4 between lactating and nonlactating human mammary cells were also documented (29). Although we cannot rule out the possibility that this observation is a consequence of the overall amount of ZnT-4 protein complex produced by the mammary cell, data from our studies of mammary cells in culture indicate that post-translational regulation of ZnT-4 localization may play a role in this process. Our results suggest that relocalization of ZnT-4 may be partially a consequence of increased cellular Zn concentration because these cells responded to Zn exposure by relocalizing ZnT-4 from the Golgi apparatus (25) to an intracellular compartment, similar to observations in human mammary epithelial cells in culture (29) and of ZnT-7 relocalization in transfected Chinese hamster ovary cells (30).
The physiologic role of ZnT-2 in maintaining Zn homeostasis in currently unknown; however, its production in the mammary gland suggests a role in mammary gland Zn metabolism. The identification of a 52-kDa glycosylated and a 45-kDa nonglycosylated form of ZnT-2 in the mammary gland concurrent with two distinct cellular localizations suggests that the presence of glycans may direct specific cellular compartmentalization as was suggested for other proteins (31,32). Furthermore, although the staining intensity of ZnT-2 at the basolateral membrane remained constant, the intensity of ZnT-2 staining at the apical membrane decreased through lactation. This decline in apical staining as lactation proceeded, perhaps as a consequence of decreasing levels of the glycosylated 52-kDa protein, provides an additional mechanistic explanation for the decreasing milk Zn concentration. Consistent with the inverse correlation between high mammary gland Zn concentration and decreased staining of ZnT-2 at the apical membrane, mammary epithelial cells exposed to physiologically high Zn levels responded by relocalizing ZnT-2 to an intracellular compartment, perhaps sequestering excess cellular Zn, as was reported by others (10).
Thus far, ZnT-1 is the only Zn transporter that has been implicated in cellular Zn export (33). Although both the mRNA expression and protein level increased through lactation, we documented the formation of distinct complexes containing ZnT-1, suggesting post-transcriptional regulation, as was predicted by others (9). Interestingly, we observed a correlation between intensity of apical staining and the intensity of the smaller 71-kDa ZnT-1–containing complex on Western blot, which was particularly high during early lactation. The decrease in apical staining and the intensity of the 71-kDa complex that occurred throughout lactation suggests that ZnT-1 may play an important role in mediating the transfer of Zn into milk during early lactation and that its contribution diminishes as lactation progresses. The results from Western blot analysis of fractions obtained by differential centrifugation further suggested that these complexes localize to different cellular compartments. Although we cannot rule out homomultimerization, it is likely that ZnT-1 participates in a heteromultimer with other currently unknown proteins. Similar to reports by others (25), we observed a relocalization of ZnT-1 in response to Zn exposure in mammary epithelial cells, from a perinuclear localization during low Zn levels, to a nonendocytotic, intracellular localization in response to high cellular Zn. Attempts to further characterize the localization in response to Zn in this cell type are currently underway.
In summary, the results from these studies indicate an association between the decline in milk Zn that occurs as lactation progresses and specific changes in gene expression and protein levels of ZnT-1, ZnT-2, ZnT-4 and Zip3 in the mammary gland of lactating rats. Furthermore, the intense staining of Zn transporters at the apical membrane of mammary epithelial cells that was observed only during early lactation was positively correlated with increased Zn transfer into milk during this period. Data from this study exemplify the functional redundancy in ZnT proteins, which may occur to help to guarantee adequate transfer of Zn into milk, thus ensuring the survival of the species. Although it appears that some of these observations may have been a result of increasing tissue Zn levels, the contribution of other lactogenic factors such as hormones (23) to this process remains to be elucidated.
|
ACKNOWLEDGMENTS |
|---|
Manuscript received 2 July 2003. Initial review completed 29 July 2003. Revision accepted 26 August 2003.
|
LITERATURE CITED |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
1. King, J. C. (2002) Enhanced zinc utilization during lactation may reduce maternal and infant zinc depletion. Am. J. Clin. Nutr. 75:2-3.
2. Moore, C.M.E., Roberto, R.D.J. & Greene, H. L. (1984) Zinc supplementation in lactating women: evidence for mammary control of zinc secretion. J. Pediatr. 105:600-602.[Medline]
3. Krebs, N. F. (1998) Zinc supplementation during lactation. Am. J. Clin. Nutr. 68:509S-512S.[Abstract]
4. Keen, C. L., Lönnerdal, B., Clegg, M. & Hurley, L. S. (1981) Developmental changes in composition of rat milk: trace elements, minerals, protein, carbohydrate and fat. J. Nutr. 111:226-236.
5. Luizzi, J. P., Bobo, J. A., Cui, L., McMahon, R. J. & Cousins, R. J. (2003) Zinc transporters 1, 2 and 4 are differentially expressed and localized in rats during pregnancy and lactation. J. Nutr. 133:342-351.
6. McMahon, R. J. & Cousins, R. J. (1998) Regulation of the zinc transporter ZnT-1 by dietary zinc. Proc. Natl. Acad. Sci. U.S.A. 95:4841-4186.
7. Kambe, T., Narita, H., Yamaguchi-Iwai, Y., Hirose, J., Amano, T., Sugiura, N., Sasaki, R., Mori, K., Iwanaga, T. & Nagao, M. (2002) Cloning and characterization of a novel mammalian zinc transporter, ZnT-5, abundantly expressed in pancreatic beta-cells. J. Biol. Chem. 277:19049-10955.
8. Kelleher, S. L. & Lönnerdal, B. (2002) Zinc transporters in the mammary gland respond to marginal zinc and vitamin A intake during lactation in rats. J. Nutr. 132:3280-3285.
9. Palmiter, R. D. & Findley, S. D. (1995) Cloning and functional characterization of a functional transporter that confers resistance to zinc. EMBO J 14:639-649.[Medline]
10. Palmiter, R. D., Cole, T. B. & Findley, S. D. (1996) ZnT-2, a mammalian protein that confers resistance to zinc by facilitating vesicular sequestration. EMBO J 15:1784-1791.[Medline]
11. Murgia, C., Vespignani, I., Cerase, J., Nobili, F. & Perozzi, G. (1999) Cloning, expression and vesicular localization of transporter Dri27/ZnT4 in intestinal tissue and cells. Am. J. Physiol. 277:G1232-G1239.
12. Gaither, L. A. & Eide, D. J. (2000) Functional expression of the human hZIP2 zinc transporter. J. Biol. Chem. 275:5560-5564.
13. Gaither, L. A. & Eide, D. J. (2001) The human ZIP1 transporter mediates zinc uptake in human K562 erythroleukemia cells. J. Biol. Chem. 276:22258-22264.
14. Cao, J., Bobo, J. A., Luizzi, J. P. & Cousins, R. J. (2001) Effects of intracellular zinc depletion on metallothionein and ZIP2 transporter expression and apoptosis. J. Leukoc. Biol. 70:559-566.
15. Milon, B., Dhermy, D., Poutney, D., Bourgois, M. & Beaumont, C. (2001) Differential subcellular localization of hZip1 in adherent and non-adherent cells. FEBS Lett 507:241-246.[Medline]
16. Dufner-Beattie, , Wang, F., Kuo, Y.-M., Gitschier, J., Eide, D. & Andrews, G. K. (2003) The acrodermatitis enteropathica gene ZIP4 encodes a tissue-specific, zinc-regulated zinc transporter in mice. J. Biol. Chem. 278:33474-33481.
17. Wang, K., Zhou, B., Kuo, Y.-M., Zemansky, J. & Gitschier, J. (2002) A novel member of a zinc transporter family is defective in acrodermatitis enteropathica. Am. J. Hum. Genet. 71:66-73.[Medline]
18. Doppler, W., Villunger, A., Jennewein, P., Brduscha, K., Groner, B. & Ball, R. K. (1991) Lactogenic hormone and cell type-specific control of whey acidic protein gene promoter in transfected mouse cells. Mol. Endocrinol. 5:1624-1632.
19. Graham, J. M. (1997) Subcellular fractionation: a practical approach. Graham, J. M. Rickwood, D. eds. Subcellular Fractionation: A Practical Approach 1997:205-242 Oxford University Press Oxford, UK .
20. Huang, L. & Gitschier, J. (1997) A novel gene involved in zinc transport is deficient in the lethal milk mouse. Nat. Genet. 17:292-297.[Medline]
21. Ackland, M. L. & Mercer, J. F. (1992) The murine mutation, lethal milk, results in production of zinc-deficient milk. J. Nutr. 122:1214-1218.
22. Moser, P. B. & Reynolds, R. D. (1983) Dietary zinc intake and zinc concentrations of plasma, erythrocytes, and breast milk in antepartum and postpartum lactating and non-lactating women: a longitudinal study. Am. J. Clin. Nutr. 38:101-108.
23. Liuzzi, J. P., Blanchard, R. K. & Cousins, R. J. (2001) Differential regulation of zinc transporter 1, 2 and 4 mRNA expression by dietary zinc in rats. J. Nutr. 131:46-52.
24. Langmade, S. J., Ravindra, R., Daniels, P. J. & Andrews, G. K. (2000) The transcription factor MTF-1 mediates metal regulation of the mouse ZnT1 gene. J. Biol. Chem. 275:34803-34809.
25. Huang, L., Kirschke, C. P. & Gitscjier, J. (2002) Functional characterization of a novel mammalian zinc transporter, ZnT-6. J. Biol. Chem. 277:26389-26395.
26. Petris, M. J. & Mercer, J.F.B. (1999) The Menkes protein (ATP7A:MNK) cycles via the plasma membrane both in basal and elevated extracellular copper using a C-terminal di-leucine endocytic signal. Hum. Mol. Genet. 8:2107-2115.
27. Petris, M. J., Smith, K., Lee, J. & Thiele, D. J. (2002) Copper-stimulated endocytosis and degradation of the human copper transporter, hCtr1. J. Biol. Chem. 278:9639-9646.
28. Michalczyk, A. A., Reiger, J., Allen, K. J., Mercer, J.F.B. & Ackland, M. L. (2000) Defective localization of the Wilson disease protein (ATP7B) in the mammary gland of the toxic milk mouse and the effects of copper supplementation. Biochem. J. 352:565-571.
29. Michalczyk, A. A., Allen, J., Blomeley, R. C. & Ackland, M. L. (2002) Constitutive expression of hZnT4 zinc transporter in human breast epithelial cells. Biochem. J. 364:105-113.[Medline]
30. Kirschke, C. P. & Huang, L. (2002) ZnT7, a novel mammalian zinc transporter, accumulates zinc in the Golgi apparatus. J. Biol. Chem. 278:4096-4102.[Medline]
31. Yeaman, C., Gall, A.H.L., Baldwin, A. N., Monlauzeur, L., Bivic, A. L. & Rodriguez-Boulan, E. (1997) The O-glycosylated stalk domain is required for apical sorting of neurotrophin receptors in polarized MDCK cells. J. Cell. Biol. 139:929-940.
32. Scheiffele, P., Peranen, J. & Simons, K. (1995) N-glycans as apical sorting signals in epithelial cells. Nature (Lond.) 378:96-98.[Medline]
33. McMahon, R. J. & Cousins, R. J. (1998) Mammalian zinc transporters. J. Nutr. 28:667-670.
This article has been cited by other articles:
|
|
 |
|
  L. Qian, V. Lopez, Y. A. Seo, and S. L. Kelleher Prolactin regulates ZNT2 expression through the JAK2/STAT5 signaling pathway in mammary cells Am J Physiol Cell Physiol, August 1, 2009; 297(2): C369 - C377. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. L. Kelleher, V. Lopez, B. Lonnerdal, J. Dufner-Beattie, and G. K. Andrews Zip3 (Slc39a3) functions in zinc reuptake from the alveolar lumen in lactating mammary gland Am J Physiol Regulatory Integrative Comp Physiol, July 1, 2009; 297(1): R194 - R201. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M.-Y. Jou, A. G. Hall, A. F. Philipps, S. L. Kelleher, and B. Lonnerdal Tissue-Specific Alterations in Zinc Transporter Expression in Intestine and Liver Reflect a Threshold for Homeostatic Compensation during Dietary Zinc Deficiency in Weanling Rats J. Nutr., May 1, 2009; 139(5): 835 - 841. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Kambe, J. Geiser, B. Lahner, D. E. Salt, and G. K. Andrews Slc39a1 to 3 (subfamily II) Zip genes in mice have unique cell-specific functions during adaptation to zinc deficiency Am J Physiol Regulatory Integrative Comp Physiol, May 1, 2008; 294(5): R1474 - R1481. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Overbeck, P. Uciechowski, M. L. Ackland, D. Ford, and L. Rink Intracellular zinc homeostasis in leukocyte subsets is regulated by different expression of zinc exporters ZnT-1 to ZnT-9 J. Leukoc. Biol., February 1, 2008; 83(2): 368 - 380. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. E. O'Brien, N. F. Krebs, J. L. Westcott, and Fang Dong Relationships Among Plasma Zinc, Plasma Prolactin, Milk Transfer, and Milk Zinc in Lactating Women J Hum Lact, May 1, 2007; 23(2): 179 - 183. [Abstract] [PDF]
|
|
|
|
 |
|
 Y. Y. Yu, C. P. Kirschke, and L. Huang Immunohistochemical Analysis of ZnT1, 4, 5, 6, and 7 in the Mouse Gastrointestinal Tract J. Histochem. Cytochem., March 1, 2007; 55(3): 223 - 234. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. L. MURILLO-FUENTES, R. ARTILLO, M. L. OJEDA, M. J. DELGADO, M. L. MURILLO, and O. CARRERAS EFFECTS OF PRENATAL OR POSTNATAL ETHANOL CONSUMPTION ON ZINC INTESTINAL ABSORPTION AND EXCRETION IN RATS Alcohol Alcohol., January 1, 2007; 42(1): 3 - 10. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Chowanadisai, B. Lonnerdal, and S. L. Kelleher Identification of a Mutation in SLC30A2 (ZnT-2) in Women with Low Milk Zinc Concentration That Results in Transient Neonatal Zinc Deficiency J. Biol. Chem., December 22, 2006; 281(51): 39699 - 39707. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. L. Kelleher and B. Lonnerdal Zinc Supplementation Reduces Iron Absorption through Age-Dependent Changes in Small Intestine Iron Transporter Expression in Suckling Rat Pups J. Nutr., May 1, 2006; 136(5): 1185 - 1191. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Elgazar, V. Razanov, M. Stoltenberg, M. Hershfinkel, M. Huleihel, Y. B. Nitzan, E. Lunenfeld, I. Sekler, and W. F. Silverman Zinc-regulating Proteins, ZnT-1, and Metallothionein I/II Are Present in Different Cell Populations in the Mouse Testis J. Histochem. Cytochem., July 1, 2005; 53(7): 905 - 912. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Dufner-Beattie, Z. L. Huang, J. Geiser, W. Xu, and G. K. Andrews Generation and Characterization of Mice Lacking the Zinc Uptake Transporter ZIP3 Mol. Cell. Biol., July 1, 2005; 25(13): 5607 - 5615. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. L. Kelleher and B. Lonnerdal Zip3 plays a major role in zinc uptake into mammary epithelial cells and is regulated by prolactin Am J Physiol Cell Physiol, May 1, 2005; 288(5): C1042 - C1047. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Huang, C. P. Kirschke, Y. Zhang, and Y. Y. Yu The ZIP7 Gene (Slc39a7) Encodes a Zinc Transporter Involved in Zinc Homeostasis of the Golgi Apparatus J. Biol. Chem., April 15, 2005; 280(15): 15456 - 15463. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. L. Kelleher and B. Lonnerdal Low Vitamin A Intake Affects Milk Iron Level and Iron Transporters in Rat Mammary Gland and Liver J. Nutr., January 1, 2005; 135(1): 27 - 32. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Wang, J. Dufner-Beattie, B.-E. Kim, M. J. Petris, G. Andrews, and D. J. Eide Zinc-stimulated Endocytosis Controls Activity of the Mouse ZIP1 and ZIP3 Zinc Uptake Transporters J. Biol. Chem., June 4, 2004; 279(23): 24631 - 24639. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
