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Food Science and Human Nutrition Department and Center for Nutritional Sciences, University of Florida, Gainesville, FL
5To whom correspondence should be addressed. E-mail: cousins{at}ufl.edu
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • transport • fetal development • lactation • gene expression
Zinc plays an important role in growth, development and reproduction. Zinc deficiency during pregnancy and lactation has adverse effects in laboratory animals, including congenital malformations, embryonic and fetal death and intrauterine growth retardation (1
). Furthermore, reduced milk production and milk zinc concentrations lead to decreased pup survival (2
). The effects of zinc deficiency in pregnant and lactating humans tend to be similar to those observed in animals (3
). Several physiological changes in maternal zinc metabolism are known to occur during pregnancy and lactation, probably to meet the special needs for zinc by the fetus and neonate. These adjustments include increased maternal intestinal zinc absorption (4
,5
), increased placental transfer of zinc to the fetus at the end of gestation (6
,7
) and higher zinc concentration in milk at the onset of lactation (8
). Zinc metabolism in fetus and pups is also developmentally regulated; for instance, in the liver of the fetal rat at near term, zinc and metallothionein concentrations are elevated (9
–11
). In addition, intestinal zinc concentrations increase drastically right after birth and then decrease steadily until initial levels are reached (10
). Finally, zinc intestinal absorption appears to be higher in suckling rats than in adult rats (12
). The underlying molecular mechanisms of these changes remain obscure.
The evolving understanding of the cellular biology of zinc homeostasis has led to identification of genes that influence influx and efflux. However, the physiological functions of these transporters are not well understood. Zinc transporter 1 (ZnT1)6 expression has been shown to be responsible for zinc efflux from cultured cells (13
). In contrast, other lines of evidence suggest that ZnT2 and ZnT4 may sequester zinc into intracellular vesicles (14
,15
). Immunohistochemical evidence clearly places ZnT1 in cellular locations that would, through cellular zinc efflux, aid in body zinc acquisition and retention (16
,17
). These sites include the basolateral membranes of rat enterocytes, renal tubular cells and the villus yolk sac of rat placenta. Similarly, ZnT4 has been localized to endosomes at the basolateral side of rat enterocytes by Murgia et al. (14
), which supports a role in zinc acquisition for this transporter. These transporters could also help coordinate fetal and neonatal zinc deposition and the maternal zinc supply necessary for these needs.
We have identified that ZnT1, 2 and 4 mRNA levels have different responses to dietary zinc (18
). Specifically, ZnT2 mRNA appears to be the most responsive to zinc nutritional status, followed by ZnT1 mRNA, whereas ZnT4 mRNA seems to be relatively refractory to differences in zinc intake. Furthermore, the tissue specificity of ZnT expression is a function of dietary zinc intake. For example, ZnT2 is not expressed in liver except when there is an acute increase in zinc intake (18
). Finally, the lethal milk (lm) mutation, which has been demonstrated by Huang and Gitschier (19
) to produce an incompletely translated ZnT4 protein (19
), yields a phenotype characterized by a lower zinc level in milk (20
,21
), suggesting that this transporter plays a critical role in determining the zinc content of milk.
Expression of these ZnTs in placenta, mammary gland and small intestine (18
) suggests that they play roles in regulating zinc homeostasis and the zinc supply during pregnancy and lactation. Initial steps to define these roles require delineation of the relative expression and tissue localization of these transporters at various stages of pregnancy/lactation and fetal/postnatal development. The results presented here show that expression of ZnT1, 2 and 4 changes markedly during pregnancy and lactation in maternal tissues and those of the neonate.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Sprague Dawley virgin, timed and untimed pregnant rats (Harlan, Indianapolis, IN) between 8 and 12 wk old were fed a commercial diet (Teklad 8604; Harlan; 60 mg Zn/kg) and tap water. Otherwise, the rats were housed under the conditions described previously (18
). Timed pregnant rats were killed at d 15 and 20 of gestation, and untimed pregnant rats were killed at d 1, 7, 14 and 20 of lactation. Rats were anesthetized with methoxyfluorane and killed by exsanguination. Liver and intestine samples were excised from fetuses at d 20 of gestation and pups at 1, 7, 14, 20 and 42 d of age. Fetuses and 1 d pups were killed by decapitation, and samples of liver and from the upper half of the small intestine were pooled from four fetuses or three pups. Maternal tissues were excised as reported previously (18
). Representative samples were also immediately fixed with buffered 4% paraformaldehyde. All procedures were approved by the University of Florida Institutional Animal Care and Use Committee.
RNA extraction and Northern analysis.
Tissues were immediately homogenized in TriPure (Roche, Indianapolis, IN); the total RNA was extracted and stored in diethyl pyrocarbonate–treated water. For Northern analysis, equal amounts of total RNA were hybridized to cDNA probes, labeled with [
-32P]dCTP using ready-to-go labeling beads (Amersham Pharmacia Biotech, Piscataway, NJ) as reported previously (17
,18
). Detection and analysis was by phosphor imaging (Storm 840 Phosphorimager; Amersham Pharmacia Biotech/Molecular Dynamics, Sunnyvale, CA). The membranes were stripped of probe and hybridized with either 18S rRNA or GAPDH (mammary gland only) for normalization.
Western analysis.
Peptides used to generate antigens were all based on data from GenBank. These were GTRPQVHSGKE (ZnT1), EDSSQQQQNPS (ZnT2) and MQLIPGSSSKWEE (ZnT4). Extensive and ongoing BLAST (http:www.ncbi.nlm.nih.gov/BLAST/) searches indicate these sequences are unique to the transporters indicated. Each was synthesized (Research Genetics, Huntsville, AL) with an additional carboxyl-end cysteine to facilitate conjugation to maleimide-activated keyhole limpet hemocyanin (Pierce, Rockford, IL). Successful synthesis was verified by amino acid analysis and mass spectrometry. The conjugated peptides were used to produce polyclonal antibodies in rabbits. Total IgG fractions were affinity purified (Sulfolink; Pierce). Specificity of these affinity-purified antibodies involved comparison of signals obtained with preimmune serum, purified IgG, affinity-purified IgG and the flowthrough effluent from affinity column chromatography as described previously (17
).
Membrane protein preparations were prepared after tissues were homogenized in HEM buffer (20 mmol/L Hepes, pH 7.4; 1 mmol/L EDTA; 300 mmol/L mannitol) with 1% protease inhibitor mixture (Sigma-Aldrich, St. Louis, MO) as described previously (17
,22
). The homogenates were centrifuged at 1000 x g for 10 min, and the postnuclear supernatant was recentrifuged at 17,000 x g for 30 min. The resulting pellet (crude membrane fraction) was suspended in HEM buffer and stored at -80°C. This membrane fraction was used for analysis of ZnT2 and ZnT4, rather than the 100,000 g total membrane fraction, to concentrate these two ZnT proteins. Analysis of ZnT1 used the total membrane fraction from the 100,000 g pellet as described previously (17
). Protein concentrations were measured (23
), and the membrane proteins (
100 µg) were resolved using 10% SDS/PAGE gels, transferred to PVDF (Immobilon; Millipore, Bedford, MA) membrane, and incubated with the specific antibodies as described (17
). Molecular mass markers were from Invitrogen (Carlsbad, CA). Visualization was with a chemiluminescence reagent (Perkin Elmer Life Sciences, Boston, MA) and X-ray film.
Immunohistochemistry.
Tissue sections were embedded in polyester wax (Polyscience, Niles, IL). Slide preparation was as described previously (22
). The affinity-purified antibodies used were described above. After incubation with the affinity-purified antibodies, immunological detection was with 3-amino-9-ethylcarbazole chromagen (AEC; Zymed Laboratories, South San Francisco, CA). Counterstaining was performed using hematoxylin. Digital micrographs were generated as described previously (22
).
Statistical analysis.
One-way ANOVA, followed by Student–Newman–Keuls multiple comparison test, was used to analyze some of the RNA data. The level of significance was set at P < 0.05. Densitometric data from the Western analysis of maternal and pup small intestine were calculated as means and pooled SEM.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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. ZnT1 mRNA levels in mammary gland tissue did not vary during gestation (Fig. 1)
. The smaller transcript of ZnT1 mRNA (Fig. 1
A) observed in other tissues (18
) was not detected in mammary gland. Levels of both ZnT2 transcripts in mammary tissue decreased significantly (P < 0.05) at the end of gestation and fell below the detection limit during lactation (Fig. 1
B). Mammary gland ZnT4 mRNA levels were constant during gestation, but decreased significantly (P < 0.05), based on normalization with 18S ribosomal RNA, by d 22 of lactation (i.e., the day after the pups were weaned) (Fig. 1
B). The amount of glandular tissue increases in the lactating mammary gland (24
) and coincides with a large increase in total RNA synthesis (25
,26
). This produces a decrease in the amount of poly A+ RNA in the total RNA population. Consequently, when normalized to GAPDH mRNA, the relative abundance of ZnT4 mRNA decreased less (by about 45%). ZnT1 and ZnT4 mRNAs exhibited a uniform abundance in the placenta at d 15 and 20 of gestation (Fig. 1)
. In comparison, placental ZnT2 mRNA levels appeared to be less, and declined between d 15 and d 20.
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), ZnT1 mRNA was maximal at d 20 of gestation (at about 1.5-fold) compared to nonpregnant rats or those at the end of lactation (d 22). ZnT2 mRNA changed to the greatest extent going from low levels in virgin females, increasing significantly (P < 0.05) during gestation, with a return to the level observed in the nonpregnant female by d 22 of lactation. By comparison, ZnT4 mRNA levels were constant during gestation, but increased modestly during lactation. In the neonatal small intestine, ZnT1 and ZnT2 mRNAs increased two- to fourfold during the suckling period compared to those levels observed in the fetus at d 20 of gestation (Fig. 2
B), and decreased after weaning (d 42). ZnT4 mRNAs increased slightly in neonates and declined slightly after weaning. ZnT1 and ZnT4 mRNA levels in fetal and pup liver increased after birth until d 20, then decreased (data not shown).
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The affinity purification of polyclonal antibody against a ZnT1 peptide was described previously (17
). Our previous experience with ZnT1, using this affinity-purified antibody, is that a single band is produced upon Western analysis. Based on migration of this band, the protein has a molecular mass of 42 and 38 kDa in membrane fractions of small intestine and liver, respectively (17
). The same antibody provided a mass of 50 kDa from membrane extracts of PC12 neuronal cells (27
). The predicted mass of ZnT1 from Swiss Prot is 55 kDa. These findings indicate the apparent mass obtained from Western analysis is less than that predicted from the amino acid sequence, and that the values obtained are tissue specific. These differences could be attributable to posttranslational modifications. Alternatively, the differences may relate to the differences in transcript sizes reported of this transporter. Two sizes in the 2- to 7-kb range are usually reported (13
,17
,28
).
Using like methods of production and affinity purification, ZnT2 and ZnT4 IgGs produced specific bands upon Western analysis of membrane fractions from maternal small intestine and mammary gland, respectively (Fig. 3
). The ZnT2 IgG produced a 28-kDa band, which is somewhat less than the molecular mass predicted from the amino acid sequence (39 kDa). The band is not found among membrane proteins from liver. The liver does not express ZnT2 mRNA (18
), further supporting the specificity of the band as being specific for ZnT2 protein (Fig. 3
A). Occasionally, a second band (85 kDa) is observed with affinity-purified ZnT2 IgG. The major (55-kDa) and minor (50-kDa) bands from the Western analysis of ZnT4 are consistent with the predicted mass of 48 kDa obtained with Swiss Prot. The 55-kDa band was used for quantitation. Flowthrough effluent from both of these affinity purifications showed no evidence of immunoreactive species (Fig. 3
A). Preincubation of both affinity-purified IgGs with a stoichiometric excess of the corresponding peptide blocked immunoreactivity (Fig. 3
B) and served as a second negative control. Preimmune serum did not show anti-ZnT2 or ZnT4 reactivity (data not shown).
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). ZnT2 levels were relatively high and constant in late gestation/early lactation, except for a reproducible decrease just before parturition, where they were similar to ZnT2 levels in nonpregnant rats (data not shown). Maternal liver ZnT1 and ZnT4 both appeared to decrease in late gestation (d 20), but increased steadily during lactation (Fig. 4
B). ZnT2 was not detected in these hepatic membrane fractions. Of particular interest is ZnT4 expression in the mammary gland (Fig. 4
C). During gestation and lactation, ZnT1 and ZnT2 are not expressed in mammary tissue at a level to allow a reasonable Western analysis.
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). Of interest is that ZnT4 levels were high at d 20 of gestation, decreased during early lactation, then increased markedly by the end of the suckling period (d 20) and following weaning (d 42). During the early suckling period, the abundance of ZnT2 was very high, and declined thereafter. In contrast, in liver, levels of both ZnT1 and ZnT4 proteins increased throughout the suckling period and after weaning (Fig. 5
B). ZnT2 protein was not detected in the liver of developing pups.
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). ZnT1 was localized in cells along the villi, appearing as punctuate-stained endosomal-like structures that were particularly abundant in those cells oriented toward the maternal blood supply (Fig. 6
A). ZnT4 was also found as punctate staining within cells of the villi and other cells constituting the entire visceral splanchnopleure (Fig. 6
B). The punctate appearance may represent vesicles where zinc is concentrated, with the ZnTs serving a sequestering function. Neither ZnT1 nor ZnT4 was found in the cells constituting the amnion. ZnT2 protein was not detected in placenta by immunostaining, despite some evidence of ZnT2 mRNA transcripts being detected in total placental RNA.
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. Intensity of the chromagen color developed with the IgG is shown in Figure 7
A. In contrast, no significant color is detected with the IgGs not retained by the affinity column (Fig. 7
B). ZnT4 appeared as an abundant protein in alveolar cells surrounding the alveolar ducts, with orientation to the area of the basal lamina as opposed to the area closer to the lumen of the duct (Fig. 7
C). In comparable sections, no ZnT2 immunoreactivity was detected in the mammary gland using affinity-purified anti-ZnT2 IgG (data not shown). Similarly, weak ZnT1 immunoreactivity was sparsely distributed at d 14 of lactation (data not shown).
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). Furthermore, considerably more immunoreactive protein is observed at d 14 of lactation (Fig. 8
C and D) than at d 1 (Fig. 8
A and B) for both transporters. At d 1, ZnT1 protein appears as punctate clusters of endosomal-like structures oriented in a unique apical position, in a line between the microvilli and the nucleus of enterocytes (Fig. 8
A). Some ZnT1 is also located apically adjacent to the brush border membrane. At d 14, the line of punctate staining is gone, staining appears less intense and is concentrated near both the apical and basolateral membranes. The distribution of ZnT4 is distinctly different. At d 1, some enterocytes are heavily stained throughout the cytoplasm. In the less heavily stained enterocytes, ZnT4 is primarily at the apical membrane, with lesser amounts at the basolateral membrane. In contrast, at d 14, ZnT4 staining is more robust overall and appears to be associated with prominent, endosomal-like structures. These are concentrated at both the brush border and basolateral membrane surfaces. At a much higher magnification of these digital images of ZnT1 and ZnT4 localization, respectively (Fig. 8
E and F), clearer views of these structures are provided.
|
. Figure 9
shows localization of these transporters at the apical surface of enterocytes along the entire villus at d 14 of lactation (Fig. 9
A and B). They do not appear to be expressed in cells of the lamina propria. Abundance at d 20 (Fig. 9
C and D) is clearly greater than d 14 of lactation. These figures also show developmental changes where enterocytes become larger and villi are longer at d 20.
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) is apical, directly adjacent to the microvilli of enterocytes lining villi. The appearance is punctate, suggestive of a vesicular association. No ZnT2 was found in crypt cells or the lamina propria. ZnT2 was limited to the duodenum, in that comparable sections of jejunum showed no detectable ZnT2 (data not shown).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Evidence that the antibodies for ZnT1, ZnT2 and ZnT4 are specific for these transporters is as follows. First, the sequences used were selected because they are unique. BLAST searches revealed no homologs to mammalian genes. Second, differential centrifugation studies revealed the immunoreactive proteins are found in membrane fractions (17,000 and 100,000 g pellets) and not in 100,000 g supernatant fractions from cell homogenates. Third, their immunoreactivity follows tissue specificity of transcript expression and abundance using Northern analysis. For example, neither ZnT2 mRNA nor ZnT2 protein is found in the liver. Fourth, their immunoreactivity is cell type specific. For example, the intestine consists of four cell types, but only enterocytes show evidence of high ZnT abundance, whereas enterochromaffin, goblet and paneth cells, and cells of the lamina propria do not. Finally, the immunoreactivity places each ZnT protein in vesicular-like structures within enterocytes.
The demonstration that the lm mutation produces a defective ZnT4 protein (19
), and the decrease in zinc secretion into milk that accompanies this murine genotype (20
,21
), places a focus of the role of this transporter on the mammary gland. The finding that ZnT4 is localized to the basal region of epithelium lining the lumen of mammary collecting ducts is consistent with the importance of ZnT4 in maintaining an adequate supply of zinc for secreted milk. In rats, the zinc concentration of milk is about 10-fold higher than in the plasma (8
); therefore, this nutrient must be concentrated in intracellular sites before being incorporated into milk. The orientation of ZnT4 in the gland is consistent with such a role for this transporter. Furthermore, yeast mutants, which highly express ZnT4, are resistant to high levels of zinc, generating the notion of an export function for this transporter (19
). The critical role of ZnT4 in zinc transfer to milk during lactation is also supported by the apparent absent or minimal ZnT1 and ZnT2 protein expression in mammary gland.
In this study, we found that the levels of ZnT4 expression, on a protein basis, increased during lactation. This finding does not follow either the higher 65Zn transfer from rat dams to pups in early lactation (29
), which gives colostrum a higher zinc content than milk for later in gestation (1
), or the reduction of milk concentration during lactation (8
). However, this apparent decrease in zinc concentration may be compensated for by the fact that the total volume of milk produced increases at least twofold during lactation (2
,30
). An analogous reduction in milk zinc concentration during lactation occurs in humans (31
), although there is an increase in glandular tissue in the lactating human breast (24
). Recent evidence suggests that ZnT4 is constitutively expressed in the human breast (24
). The apparent discrepancy between our results with mammary ZnT4 in rats showing increased expression during lactation and constitutive expression in humans (24
), despite the obvious species differences, could be related to the basis of normalization. The bases for these were total membrane protein, as reported here (Fig. 4)
, or a specific protein (ß-actin) for the human samples. The results shown in the present report support our previous findings in rats, where mammary ZnT4 expression was refractory to changes in zinc intake (18
). This could explain why zinc supplementation does not increase the zinc concentration of human milk. Furthermore, if milk zinc levels are regulated primarily by one transporter, the low zinc concentration of human milk in some individuals (32
) could be explained by genotypic differences in the transporter.
Total zinc concentrations in the fetus do not change appreciably during gestation, but, in contrast, the rates of zinc accumulation by the fetus increase with gestational age (10
,11
). This clearly places a focus on the zinc transport capacity of the placental feto-maternal interfaces. In rats, half of the total fetal zinc is acquired in the last trimester (33
). Developmental defects associated with maternal zinc deficiency further emphasize the importance of placental zinc transport (1
,34
). Our results clearly show that ZnT1 and ZnT4 are localized to the placental region, where fetal blood and maternal blood circulate, separated by endothelial and other cells forming the interface where nutrient transport occurs. A null mutation of the ZnT1 gene produces embryonic lethality (28
), demonstrating the potential importance of this transporter during development. Previously, we showed that ZnT1 is sensitive to dietary zinc intake, whereas ZnT4 expression is quite refractory to intake (18
). The important finding that the metal response element-binding transcription factor-1 (MTF-1) is required for ZnT1 gene regulation in the murine visceral yolk sac (28
) supports the observed influence of zinc from the dietary supply on expression of this and other zinc-regulated transporters. A null mutation of the MTF-1 gene produces embryonic lethality at d 14 of gestation (35
). Consequently, embryonic lethality would be expected for other MTF-1–regulated genes including MTF-1–regulated zinc transporters, such as ZnT1, that influence maternal/fetal zinc homeostasis.
The level of ZnT2 protein in neonatal small intestine seems to peak in early lactation. ZnT2 mRNA is heavily dependent on dietary zinc regulation in small intestine and kidney (18
). The higher ZnT2 expression observed in neonatal pups could be a consequence of expression induced by the increased levels of zinc in milk immediately after parturition (33
). ZnT2 has the capacity of concentrating zinc in endosomal acidic vesicles in cultured hamster kidney cells, which markedly increases their viability in high zinc medium (15
). In the intestine of the newborn, these vesicles could act as a zinc reserve, a protective mechanism, or be a reflection of a high rate of zinc flux through enterocytes of neonates.
As shown in this report, the relative levels of mRNA and protein for a specific ZnT do not always follow closely. Both are likely to be regulated in a zinc-dependent fashion, but, because so little is known about the control points involved and their interrelationship to known and yet unidentified transporters, any attempt to explain linkages at this time would be purely speculative.
Roles for ZnTs in intestinal zinc absorption are still obscure. The localizations we have observed in intestinal ZnTs during pregnancy and lactation may provide some clues as to their relative importance. Available data suggest zinc absorption increases during these physiologic conditions (4
,5
). Considering the maternal gut hypertrophy that occurs during lactation (36
), the same relative rates of transporter protein synthesis would yield a greater absorptive capacity. Immunohistochemistry shows both maternal ZnT1 and ZnT4 have an apical distribution at d 1 of lactation compared to d 14, where they appear localized to both apical and basolateral regions. These findings agree with our previous studies on ZnT1 in adult male rats, where both apical and basolateral regions were sites of immunologically reactive ZnT1 (17
). However, the unique abundance of ZnT1 in the maternal small intestine at d 1 of lactation, appearing as a line of endosomal-like structures, suggests this is a storage form of zinc used for maternal needs, including lactation. The apical/microvillar orientation of ZnT2 is also unique and may reflect a specialized route of transcellular zinc movement, storage or secretion to the intestinal lumen. The different intracellular localization of ZnT proteins in the small intestine may relate to a functional redundancy among these transporters. Multiple transporters acting in a tissue-specific manner may explain why the dysfunctional ZnT4 gene in lm mice does not yield altered intestinal absorption of zinc, but does reduce zinc secretion into milk by the mammary gland. This could potentially be explained by the low ZnT1 or ZnT2 expression in that organ compared to the intestine.
Both ZnT1 and ZnT4 appear to be developmentally upregulated in neonatal intestine. The high levels of ZnT1 and ZnT4 observed in the d 20 fetal small intestine could indicate that prenatal expression allows for fetal zinc acquisition from swallowed amniotic fluid, which was shown to be important for carbohydrate, amino acid and protein use by the developing fetus (37
). Our results support the upregulation of ZnT4 in the neonatal intestine originally identified by Murgia et al. (14
). Recently, attention focused on the ZnT4 gene as a candidate to account for the human autosomal recessive mutation that produces the zinc malabsorption disorder Acrodermatitis Enteropathica (38
,39
). Furthermore, identification of single-nucleotide polymorphisms in the human ZnT4 gene (38
) could lead to the identification of individuals with differing abilities to transport zinc. However, recently, mutations in the ZIP4 gene have been identified as the defect producing Acrodermatitis Enteropathica (40
). This previously unidentified gene clearly must produce a transporter of major importance for zinc absorption by humans. Rodent homologs will certainly be identified, and may be of equal importance for zinc absorption in these species. As such new information emerges, a challenge will be to identify the quantitative role for each ZnT transporter in absorption and retention.
The molecular events associated with zinc transporter gene expression are also complex, given that two transcript sizes are found for most ZnT mRNAs and at least one ZIP mRNA (14
,17
,18
,24
,28
,40
–44
), for which there are a number of explanations. They include difficulty in establishing start sites (15
), and the potential for differential functions of the proteins these transcripts yield. An example is the exact sequence homology between a segment of the hZTL1 gene, a human zinc transporter of large molecular mass and hZnT5, a much smaller transporter (41
,42
). Most likely, differential processing of the initial transcripts accounts for the difference. Similarly, posttranslational modifications and other events influence the apparent size of the ZnT transporters. For example, molecular mass estimates from Western analysis of transporter proteins vary among tissues and do not closely follow masses predicted from amino acid sequence data. An explanation for this is related to differences in glycosylation. An example of such an effect of glycosylation has been shown recently for the divalent metal transporter DMT1 in rat duodenum (45
) and ZnT4 in human breast tissue (24
).
The emerging view of zinc transport shows involvement of an array of transporter proteins from at least two identified gene families. There are at least four members of the ZIP family and six members of the ZnT family. There appears to be a hierarchy involved because mutations of two genes, the murine ZnT4 and the human ZIP4 gene, lead to phenotypes with documented pathology (19
,40
). Furthermore, as more information is acquired, unanticipated roles for zinc transporters [e.g., ZnT5’s role in osteoblast maturation and cardiac electrical conductance (44
)] may continue to emerge.
The data presented in this report show differential expression of ZnT1, ZnT2 and ZnT4 genes during gestation, lactation and fetal/neonatal development. The transporter localization data suggest intracellular compartmentalization is responsive to changes in organ proliferation and cellular development, which may reflect nutritional requirements for zinc during development, and hence suggest the possibility of hormonal regulation of ZnT gene expression. The zinc transporters studied in these experiments are believed to have an exporter function and at least two of the genes, ZnT1 and ZnT2, are zinc regulated. Further experiments are needed to place these and the expanding array of zinc transporters within a functional, teleological context.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Present address: Instituto de Biologia Experimental, Universidad Central de Venezuela, Caracas, Venezuela.
3 Present address: Mayo Clinic, Jacksonville, FL.
4 Present address: Mead Johnson Nutritionals, Evansville, IN.
6 Abbreviations: Da, Dalton; IgG, immunoglobulin G; ZnT1, zinc transporter 1; ZnT2, zinc transporter 2; ZnT4, zinc transporter 4.
Manuscript received 30 August 2002. Initial review completed 2 October 2002. Revision accepted 24 October 2002.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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3 individual animals or 3 pooled samples from three or four fetuses or pups). Values are significantly different from corresponding nonpregnant dams (A) and 20 d fetal (B) samples at *P < 0.05 or **P <0.01.
