Zinc (Zn) is an essential component of many proteins and enzymes (1–3). The important functions of Zn in growth and development (4–6) result in relatively high requirements of Zn in infants. The observation of higher content of milk Zn during early lactation suggests that Zn requirements are high during the neonatal period (7). Term newborns are born with large hepatic Zn stores, but these reserves are considerably lower in infants born prematurely (8). Formulas for preterm infants often have higher levels of Zn than those for term infants, and breast milk given to preterm infants is usually fortified with Zn to a total concentration of 3.7 mg/L (9); however, physiological consequences and optimal timing of Zn supplementation are still unknown, and are of concern with regard to not only alleviating Zn deficiency but also inducing potential toxic effects from excess Zn exposure. It is suggested that neonatal Zn requirements vary with postnatal maturity and are different from those in adults (8), but it is unclear how regulation of neonatal Zn homeostasis is modulated.
In adults, Zn homeostasis is achieved principally through a balance between intestinal absorption and endogenous excretion (10). Fractional Zn absorption is inversely related to dietary Zn intake, whereas intestinal absorptive capacity is high at low levels of Zn intake. During periods of higher levels of Zn intake, there is a decrease in absorptive efficiency but an increase in the total amount of Zn absorbed. However, an upper limit exists where increasing Zn intake >20 mg in solution does not result in greater Zn absorption (11). In contrast, in infants, the size of the exchangeable Zn pool (an indicator of Zn status) was found to be positively correlated to levels of dietary Zn (12). This is interpreted to suggest that counterintuitively, Zn absorption in infants is higher in response to increased dietary Zn intake, whereas Zn absorption in adults is limited and regulated. Furthermore, fractional Zn absorption in adults is negatively correlated with dietary Zn intake under the dosage ceiling, but this relation is missing in infants (12), suggesting that the capacity of Zn absorption in infants is not as tightly regulated as in adults, and thus the high Zn absorptive capacity in infants may induce potential toxicity. As a result, we hypothesized that mechanisms regulating Zn homeostasis are developmentally regulated and are dependent on intestinal maturation.
Zn homeostasis is primarily regulated via intestinal Zn transport proteins (13,14), and 2 families of Zn transporters have been identified that participate in the regulation of Zn homeostasis: zinc-regulated transporter (ZRT), iron-regulated transporter (IRT)–like proteins (ZIP), and cation diffusion facilitator (CDF). The ZIP family imports Zn into the cytosol, either directly across the plasma membrane or from intracellular vesicles (15,16), whereas the CDF family functions to export Zn. The primary regulator of intestinal Zn import is Zip4 and mutations in the Zip4 gene are responsible for acrodermatitis enteropathica (17), a rare genetic disorder in humans that results in severe Zn deficiency from impaired Zn absorption. Zip4 protein is localized intracellularly under Zn-adequate conditions but becomes associated with the apical membrane during severe Zn deficiency (18). Zip1 and Zip3 are also expressed on the plasma membrane and are responsible for intracellular Zn accumulation (15,19). Overexpression of Zip1 and Zip3 in cells has been shown to increase Zn uptake in several cell lines (20,21).
In contrast, ZnT1 is a member of the CDF family that is localized to both apical and basolateral membranes of the small intestine, and is responsible for Zn efflux from the cytosol (18,22). ZnT1-transfected cells increase Zn efflux and reduce the intracellular Zn concentration, suggesting that ZnT1 can confer resistance to cellular Zn toxicity (23). ZnT2 is localized to intracellular compartments and sequesters Zn into intracellular vesicles for storage (18,24,25). Furthermore, metallothionein-1 (MT-1) is a metal-binding cytoplasmic protein, which binds intracellular Zn with high affinity and may function as a biologically important reservoir of Zn in a number of organs under Zn-limiting conditions (26,27).
In the present study, cultured human intestinal cells of different degrees of maturity suggestive of enterocytes from preterm, newborn, and adults were used to investigate the development of Zn absorption mechanisms. Although other cell types in the intestine (eg, goblet cells) produce mucin, which has been indicated to affect iron uptake (28), the effect on Zn uptake by enterocytes is not known. Caco-2 cells can be cultured to differentiate into polarized monolayers with tight barriers, which express characteristics of intestinal cells (29). Importantly, Caco-2 cells differentiate at rates proportional to increasing culture time (30,31). Thus, we used undifferentiated Caco-2 cells (U) as a model of the developing neonatal intestine and differentiated polarized Caco-2 cells (D) as a model of mature intestinal cells. Finally, human fetal intestinal cells (FHs 74 Int, F) do not form polarized monolayers in culture, and are genotypically more similar to immature enterocytes in preterm infants (32). In the studies presented below, we tested the hypothesis that the balance between Zn uptake and secretion is dependent on enterocyte differentiation, which in turn affects Zn transporter regulation.
MATERIALS AND METHODS
Caco-2 cells (HTB-37, American Type Tissue Culture Collection, Rockville, MD) were used between passages 30 and 40 and cultured in minimal essential medium (Invitrogen Life Sciences, Rockville, MD), containing 10% fetal bovine serum, 1% penicillin, and streptomycin (10 U/mL and 1 mg/mL, respectively) at 37°C with 5% CO2. U cells were cultured for 4 days (before confluence) and then studied. D cells were used at day 12 after seeding (postconfluence). F cells (CCL-241, American Type Tissue Culture Collection) were used between subculture 10 and 20, and cultured in hybricare medium ATCC 46-X (American Type Tissue Culture Collection) supplemented with 30 ng/mL epidermal growth factor (Sigma, St Louis, MO), 10% fetal bovine serum, and 1% penicillin and streptomycin (10 U/mL and 1 mg/mL, respectively) at 37°C with 5% CO2 and 95% humidity atmosphere.
Cell Viability Assay
Cells were seeded into 96-well plates at a density of 5 × 104 cells/cm2 and incubated in an atmosphere of 5% CO2 at 37°C for 24 hours. Confluent cells were treated with ZnSO4 (0, 10, 25, 50, 100, 150, 300, and 750 μg/mL) containing serum-free medium for 24 hours, and cell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Roche Applied Science, Mannheim, Germany) based on the ability of living cells to convert dissolved MTT to insoluble formazan using the manufacturer's protocol.
To determine effects of Zn exposure on Zn uptake, F, U, and D cells were cultured on polycarbonate plates in growth medium and treated with serum-free medium containing 65Zn (<0.1 μCi/well), which was supplemented with 50 μmol/L ZnSO4. Cells were incubated at 37°C for 15 minutes. After being washed 3 times with ice-cold phosphate-buffered saline (PBS) containing 1 mmol/L ethylenediaminetetraacetic acid (EDTA), cells were solubilized with 1 mol/L NaOH and Zn uptake was determined by quantifying 65Zn in the cell fraction using a gamma counter. Uptake of 65Zn in F and U cells was normalized to cell number, and cellular protein content (quantified as specified below) was used to normalize between U and D cells.
Quantification of Zn Transporter Gene Expression by Real-time RT-PCR
Total RNA was extracted from cells with Trizol (Invitrogen, Carlsbad, CA) using the manufacturer's protocol. Integrity of RNA was verified following electrophoresis through 1% agarose and ethidium bromide staining. Total RNA (1 μg) was reverse transcribed using Taqman reverse transcription kit (Applied Biosystems, Foster City, CA). The reverse transcription reaction was performed at 48°C for 30 minutes followed by 95°C for 5 minutes. The following gene-specific primers for Zip1, Zip3, Zip4, ZnT1, ZnT2, ZnT5, MT-1, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were designed using Primer Express software (Applied Biosystems) and purchased from Qiagen (Valencia, CA): Zip1 (forward, 5′-GGCCATGGGCTTCTTCCT; reverse, 5′-GGCCCTGACTGCTCCTTGTA-3′), Zip3 (forward, 5′-TTCTGGCCACGTGCTTCAA; reverse, 5′-GCCGAGGCTCAGGACCTT-3′), Zip4 (forward, 5′-CCAGTGTGTGGGACACGGTAT; reverse, 5′- TGTTCCGACAGTCCATATGCA-3′), ZnT1 (forward, 5′-GAAGAAGATAGGGCTGGACAACTT; reverse, 5′- CCCAAGGCATCTCCAAGGA-3′), ZnT2 (forward, 5′-TGCATGGAGGCCAAGGA; reverse, 5′-CCGTGTATGACCGGATTGC-3′), ZnT5 (forward, 5′-CAGCCATTCACACCATATGCAT; reverse, 5′-ACACCCCTCATGTTAGCATTCA-3′), MT-1 (forward, 5′-CCGGACCAACTCAGACTCTTG; reverse, 5′-TTCACATGCTCGGTAGAAAACG-3′), GAPDH (forward, 5′-CCACCCATGGCAAATTCC; reverse, 5′-TGGGATTTCCATTGATGACAAG-3′), Quantitation of Zip1, -3, -4, ZnT1, -2, and MT-1 mRNA levels using real-time polymerase chain reaction (PCR) was performed using the ABI 7900HT real-time thermocycler with SYBR Green technology (Applied Biosystems) as previously described (33). Each sample was analyzed in duplicate and normalized to GAPDH using the following equation: fold change = 2(Ct Gene – Ct GAPDH).
Peptide fragments for Zip4 (CAEETPELLNPETRRL) were synthesized by Genemed Synthesis Inc (San Francisco, CA), and sequences were verified by amino acid analysis and mass spectrometry. These fragments were conjugated to hemocyanin and used to generate Zip4 antisera in rabbits. The ZnT1 antibody was produced as previously described (34). Specificity of peptide antisera was verified by the reduced appearance of unique immunoreactive bands after co-incubation with peptide (1 mg) and the appearance of specific bands not detected on a Western blot of Caco-2 cell proteins after incubation with preimmune serum (Fig. 1). Immunoreactive bands of Zip4 and ZnT1 were identified at ∼60 kDa (Fig. 1A) and ∼47 kDa (Fig. 1B), which were also the bands detected on the cellular membrane by biotinylation assay.
For protein isolation, cells were harvested from 10-cm dishes in 20 mmol/L HEPES pH 7.4, 1 mmol/L EDTA, and 250 mmol/L sucrose, with protease inhibitors (Sigma). Samples were sonicated at 30 kHz for 15 seconds twice (Cell Disruptor 185, Branson Sonic Power, Danbury, CT) and centrifuged at 800g for 15 minutes at 4°C, and the postnuclear supernatant was transferred to new tubes. Protein concentration was determined by the Bradford assay (Bio-Rad, Hercules, CA). Proteins (100 μg) were electrophoresed through polyacrylamide gels (10%), transferred onto nitrocellulose membrane at 350 mA for 60 minutes, and blocked overnight in PBS + 0.1% Tween-20 (PBST) with 5% nonfat milk at 4°C. Consequently, blots were incubated with purified antibodies (ZnT1, Zip4, and ZnT2, 1 μg/mL in PBST) for 45 minutes, washed 3 times with PBST, and detected with donkey anti-rabbit IgG-horseradish peroxidase (Zip4, ZnT2, and ZnT1, 1:25,000 in 5% milk for 45 minutes). After a 30-minute wash with PBST, bands were detected using Super Signal Femto Chemiluminescent Reagent (Pierce, Rockford, IL), and molecular mass of visualized proteins was assessed relative to standard full-range rainbow molecular weight markers (GE Healthcare, Piscataway, NJ).
Detection of Zip4 and ZnT1 at the Plasma Membrane by Cell Surface Biotinylation
To determine the effects of Zn exposure on abundance of Zip4 and ZnT1 at the plasma membrane, confluent U and D cells were treated in serum-free medium with 50 μmol/L Zn for 1 hour at 37°C; then cell surface proteins were biotinylated with N-hydroxysulfosuccinimide sodium salt biotin (Pierce, 0.5 mg/mL) at 4°C for 1 hour. Cells were washed twice with 50 mmol/L glycine (pH 5) followed by 3 washes with ice-cold PBS, scraped into cold lysis buffer (50 mmol/L Tris-HCl pH 7.4, 2 mmol/L EDTA, 2 mmol/L ethylene glycol tetraacetic acid, plus protease inhibitors) and sonicated for 30 seconds on ice. The crude membrane fraction was pelleted by ultracentrifugation at 150,000g for 30 minutes and resuspended in lysis buffer containing NaCl (0.1 mol/L). Membranes were solubilized with sodium dodecyl sulfate (SDS; final concentration to 0.2%) at 60°C for 5 minutes; then Triton X-100 was added (final to 1%) and briefly sonicated on ice. Insoluble material was pelleted by ultracentrifugation at 100,000g for 20 minutes, and supernatant was incubated with 50 μL of a 1:1 slurry of Ultralink-neutravidin beads (Pierce) while rocking at room temperature for 1 hour. Beads were pelleted by centrifugation at 2300g for 1 minute and washed 4 times with PBS + 1% Triton X-100. Biotinylated proteins were eluted by boiling in SDS-polyacrylamide gel electrophoresis buffer containing mercaptoethanol (5%) and immunoblotted for Zip4 and ZnT1 as described above. Cells not treated with biotin were used to assess nonspecific binding of proteins to Ultralink beads.
Comparisons between F, U, and D cells were analyzed by 1-way ANOVA and posttested by Tukey test using Prism 3.0 (GraphPad Software, San Diego, CA). Statistical comparisons between −Zn and +Zn were made using the Student t test. All of the data are represented as mean ± SD, and significant effects of treatments were determined at P < 0.05.
Effects of Enterocyte Phenotype on Resistance to Exogenous Zinc
To compare the relative viability of fetal and adult intestinal cells exposed to Zn, cells were cultured and treated with serum-free medium containing different levels of Zn for 24 hours, and lethal dose (LD)50 concentrations (Zn concentration resulting in a 50% reduction in cell viability) were determined by the MTT assay (as a measure of Zn resistance). The LD50 were 67.9 ± 5.3, 117.0 ± 5.2, and 224.4 ± 3.7 μmol/L Zn for F, U, and D cells, respectively (Fig. 2). Viability of F cells (77.6 ± 11.5, P < 0.05) was significantly different from that of U and D cells (92.9 ± 3.4 and 95.7 ± 8.9) at 50 μmol/L Zn and at higher concentrations. U cells had significantly lower viability (25.6 ± 5.1, P < 0.05) than D cells (58.6 ± 3.1) at 150 μmol/L Zn. Thus, our data indicate that F cells are more intolerant to Zn exposure than adult cells, and that Zn resistance increases with intestinal maturity.
Effects of Enterocyte Phenotype on 65Zn Uptake
Because Zn resistance was different between fetal and adult cells, Zn uptake was measured with 65Zn to examine the degree to which Zn is imported into the cells (Fig. 3). Using cell number as a normalization factor, Zn uptake by F cells was higher (∼3-fold) compared with U cells. Because Caco-2 cell proliferation is arrested during differentiation, U and D cells were normalized with regard to total protein levels instead of cell number. Our data indicate that Zn uptake was higher (∼3-fold) by U cells compared with D cells. Taken together, these results show that fetal cells have a higher capacity for Zn uptake and that this capacity decreases with intestinal maturation.
Effects of Enterocyte Phenotype on Relative Expression of Zn Transporters
To determine differences in expression of Zn transporters and MT-1 between cell models, relative mRNA expression was measured in F, U, and D cells using real-time PCR. In F cells, Zip4 mRNA expression was undetectable; expression of Zip3 and MT-1 mRNA was significantly higher in F cells than in U and D cells (Fig. 4). Zip4 expression was higher (∼3-fold) in the more mature D cells compared with U cells (Fig. 4). The relative expression of ZnT1 was 5-fold and 7-fold higher in U cells and D cells, respectively, compared with F cells, and similar results were observed when ZnT2 mRNA expression was examined (U, 2-fold; D, 9-fold of F cells).
Effects of Zn Exposure on Zn Transporter Expression in F Cells
Zn significantly increased ZnT1 mRNA expression and protein abundance in F cells (Fig. 5A and B). Zn exposure also significantly increased ZnT2 mRNA expression in F cells (Fig. 6A), but without changes in ZnT2 protein levels (data not shown). Interestingly, we found that Zn exposure dramatically increased MT-1 mRNA level only in F cells (Fig. 6B).
Effects of Zn Exposure on Zn Transporter Expression and Localization in U and D Cells
Zn exposure significantly increased ZnT1 mRNA expression in D cells (P < 0.05) (Fig. 5A) and ZnT1 protein abundance, to some extent, in both U cells (P = 0.10) and D cells (P = 0.06) (Fig. 5B). Zn induced ZnT2 mRNA levels in both U and D cells (Fig. 6A) without significantly changing protein levels (data not shown). Zip4 mRNA expression in U and D cells was not affected by Zn treatment (Fig. 7A), whereas Zip4 protein level increased significantly with Zn exposure in U cells but decreased in D cells (Fig. 7B).
To elucidate the discrepancy of Zip4 protein responses to Zn between U and D cells, a biotinylation assay was then used to quantify changes in ZnT1 and Zip4 localization. Zn exposure increased membrane-associated ZnT1 protein level (2.3-fold) only in D cells (Fig. 8A). Zip4 membrane-associated protein abundance was significantly decreased by Zn only in D cells (Fig. 8B). Thus, it is likely that Zn exposure decreased Zn import into D cells by lowered Zip4 protein abundance and increased endocytosis of Zip4 from plasma membrane, whereas increased Zip4 protein was not functionally expressed on the plasma membrane in U cells.
Zn absorption in healthy adults is inversely related to the dose of dietary Zn (11), and this regulatory process in adults appears to be achieved over a 10-fold change in Zn intake through the synergistic regulation of Zn absorption and endogenous excretion (35). The regulation of Zn absorption and excretion has been reported to be different between infants and adults (10,12). In premature infants fed breast milk, urinary Zn excretion is considerably higher than in adults (36), suggesting that dietary Zn absorption is greater when the intestine is immature relative to that in the adult. These observations further suggest that in early life, intestinal Zn absorption is not as tightly regulated as in adults. We found that although cellular uptake of 65Zn was markedly greater in F cells, fetal cells were also much more intolerant to Zn exposure than were U or D Caco-2 cells. Our data suggest that differential expression of Zn transporters and their divergent response to Zn exposure in these cells were responsible for these differences in the regulation of Zn homeostasis (Fig. 9). These observations are in support of our hypothesis that cellular regulation of Zn absorption is developmentally regulated (12) and that the high capacity to absorb Zn in infants may induce potential toxicity. The timing and dose of Zn supplements given to preterm infants may need to be evaluated further.
Intestinal uptake of Zn is regulated predominantly via Zn transporters (Zip and ZnT proteins) (18). Although changes in Zip4 localization and expression are believed to be the primary regulators of intestinal Zn absorption (37,38), in humans and animal models, Zip4 is not the only Zn transporter responsible for Zn import because other Zn transporters also participate in absorption of dietary Zn (39). In fact, Zip4 was not detected in fetal cells, and the absence of Zip4 expression in these cells was accompanied by higher Zip1 and particularly Zip3 expression relative to U and D Caco-2 cells. Unlike the tight regulation of Zip4 found in Zn-deficient mice, Zip1 expression was not affected in mice fed a Zn-deficient diet for 5 days (40), and expression level of Zip1 and Zip3 is not regulated at the transcriptional level (16,41). Instead, Zip1 and Zip3 are tightly regulated posttranslationally because results from a transfected mouse cell model show that intracellularly distributed Zip1 and Zip3 transporters cycle rapidly to and from the plasma membrane in Zn-replete cells at a slower rate compared with Zip4 (42). These results suggest that intestinal regulation of Zip1 and Zip3 is considerably less sensitive than that of Zip4, which may be responsible for our observation of less regulation of Zn uptake in fetal cells as compared with mature enterocytes.
Although ZnT1 expression was lower in fetal cells than in U and D cells, Zn exposure in fetal cells significantly increased ZnT1 expression. It has been shown in rats that Zn supplementation increases ZnT1 mRNA and protein levels in the intestine (43), presumably through functional changes in metal-response element-binding transcription factor-1 (MTF-1). MTF-1 functions as a cellular Zn sensor because the affinity of MTF-1 to MTF-1-responsive promoters is enhanced following binding of Zn to its Zn finger domain (44). MTF-1, once bound to Zn, translocates to the nucleus and increases the transcription of downstream genes, including ZnT1 and MT-1. These findings suggest that the expression of ZnT1 and MT-1 in fetal cells may be transcriptionally regulated upon Zn exposure with increased binding of intracellular Zn to MTF-1.
In addition, the expression of ZnT2, a Zn sequestration protein, increased with maturity, suggesting that fetal enterocytes are not capable of sequestering Zn in intracellular compartments as well as mature enterocytes. ZnT2 facilitates the accumulation of Zn in intracellular organelles (24), and intestinal ZnT2 mRNA levels have been shown to be upregulated with Zn supplementation in rats (45). Zn exposure increased ZnT2 mRNA expression without affecting ZnT2 protein levels in our cell models. Although this may be due to the short time period we used for Zn exposure, our data suggest it may also reflect a hierarchy of regulatory response such that cellular Zn export through ZnT1 is the primary point of regulatory control. Zn-induced expression of ZnT2 paralleled higher MT-1 expression in fetal cells, possibly protecting against Zn toxicity through Zn binding (46). We hypothesize that fetal cells respond to Zn exposure by inducing expression of ZnT1 and MT-1, thereby attempting to achieve Zn homeostasis; however, tolerance to Zn is limited as shown by the lower LD50 levels, possibly because of a limited capacity to sequester Zn into intracellular compartments through mechanisms such as ZnT2.
In contrast to fetal cells, both U and D Caco-2 cells expressed Zip4 transcripts as well as those of Zip1, Zip3, ZnT1, and ZnT2. Expression of Zip4, ZnT1, and ZnT2 increased with advancing maturity, whereas Zip1 and Zip3 expression declined, suggesting that differential expression of uptake and export mechanisms are developmentally regulated. ZnT5 has been recognized as a bidirectional Zn transporter in human enterocytes, participating in not only Zn efflux but also Zn uptake (47,48). In fact, mRNA expression of ZnT5 was observed in U cells (data not shown). These results suggest that Zip1, Zip3, and ZnT5 may participate in Zn uptake in U cells, but are not appropriately regulated by Zn exposure as shown by high 65Zn uptake. In more mature cells, Zip4, ZnT1, and ZnT2 appeared more sensitive to Zn exposure and are likely to be significant regulators.
In mice, Zip4 is localized at the apical membrane of enterocytes (49), and the ability of Zn to stimulate endocytosis of Zip4 has also been shown in transfected cells (38,50), suggesting that Zn-regulated trafficking of Zip4 is a key mechanism of cellular Zn homeostasis. The observation that intestinal Zn absorption in mice is depressed upon intraperitoneal injection of large doses of Zn reflects the Zn-dependent regulation of Zip4 expression and localization. In U cells, Zn exposure increased total Zip4 protein, but it was not functionally expressed on the membrane. With increasing Zip4 expression in the D cells, Zn exposure decreased Zn import into cells by producing less Zip4 protein and increasing endocytosis of Zip4 from the apical membrane. We speculate that Zn homeostasis during development is partially achieved through posttranslational regulation of Zip4 expression. Furthermore, Zn supplementation of Caco-2 cells has been shown to result in increased ZnT1 mRNA and protein levels (51), and we also found that ZnT1 expression was induced by Zn exposure in U cells, and translocated to the apical membrane of the D cells. This suggests that ZnT1 is transcriptionally and posttranslationally regulated, and that regulation of its expression and localization depends on intestinal maturation.
In conclusion, our data indicate that Zn absorption is developmentally regulated, and the developmental maturation of intestinal Zn sequestration, efflux, and import mechanisms may be responsible for different abilities of infants and adults to regulate Zn absorption. Although it is currently not understood whether these observations can be recapitulated in animal models or human infants, the implications of ontogenetic regulation of Zn absorption mechanisms must be taken into consideration when proposing and implementing Zn intervention programs for the target population, including infants.
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