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Nutritional Genomics Laboratory, Food Science and Human Nutrition Department and Center for Nutritional Sciences, University of Florida, Gainesville, FL 32611-0370
3 To whom correspondence should be addressed. E-mail: cousins{at}ufl.edu.
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ABSTRACT |
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 TOP ABSTRACT LITERATURE CITED |
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KEY WORDS: • zinc • gene regulation • transporters • metabolism • genomics
The pace of biological research has a tremendous impact on the nutritional sciences such as our understanding of how trace elements maintain the health of animals including humans. Human and murine sequencing initiatives have broadened the opportunities available. The senior author was asked to present at the Trace Elements in Man and Animals–9 meeting a view of future technologies that would be applicable to the field. Techniques for defining target genes influenced by intake of a specific trace element micronutrient, namely, zinc, were particularly highlighted (1). At the time, differential mRNA display was emphasized as a major tool for examining differentially regulated genes. Subsequently, cDNA array analysis evolved as a primary technique for genome mining to identify physiologically relevant differentially regulated genes (2).
Genes regulated by zinc
Differential display and cDNA arrays are used for the assessment of genes that are regulated by the dietary zinc supply. Both technologies have inherent advantages and disadvantages, and the literature is replete with descriptions of these. For research on the phenotypic outcomes of altered trace element status, differential display has the advantage of being unlimited because cDNA sequences are generated; this is in comparison to cDNA arrays, which are usually proprietary and have finite genetic information imprinted. In addition, with differential display, more than two treatments, e.g., multiple levels of a trace element, can be simultaneously compared (3– 5). The method is technically challenging and labor intensive. In comparison, with cDNA arrays, thousands of sequences including control genes used for normalization, etc. are examined simultaneously, and it is impractical to compare more than two treatments (2). Our experiments to define the genes that are responsive to zinc restriction and excess used both technologies, and this experience led us to the conclusion that for experiments dealing with nutrition, both methods have advantages and disadvantages. Both require independent confirmation at the transcriptome level [usually quantitative real-time polymerase chain reaction (Q-PCR) 6 or Northern analysis] and if possible also at the protein level.
Rat small intestine. The small intestine of the rat, which is integral to zinc homeostasis and is a site of dysfunction upon zinc deficiency, was the target for our first differential display experiments (3). A standard protocol was used whereby growing male rats were fed a zinc-deficient diet. A genetic marker of zinc deficiency includes reduced kidney metallothionein (MT) mRNA levels. Of immediate interest were the findings that 1) transcript abundance for the vast majority of genes expressed was not affected by zinc deficiency, and 2) more than half of the genes identified as differentially expressed in zinc deficiency were upregulated. The former finding was counter to the dogma that zinc deficiency would produce a universal reduction in gene expression through reduction in RNA polymerase activity, structural alterations to chromatin or other zinc-dependent factors. Upregulation of some genes suggested a compensatory mechanism for loss or reduction of function. A number of the genes confirmed as differentially regulated can only be characterized as expressed sequence tags (EST). Although not immediately useful for linking genetic information to phenotypic situations that are associated with zinc nutrition, ESTs provide genetic sequence data that can be used later with expanding genome databases including that for the rat, which is currently less complete than the murine or human databases.
Of particular interest in differential display data were two known differentially expressed genes that could be placed within the context of zinc deficiency. These were cholecystokinin and uroguanylin (UG), which were both upregulated and could be related to decreased food intake and diarrhea, respectively, associated with the deficiency (2, 6). We chose to further explore the UG upregulation. UG is a cysteine-rich hormone that influences intestinal fluid balance through the guanylate cyclase-C pathway (7). Its upregulation is believed to produce outcomes comparable to the secretory diarrhea that is stimulated by heat-stable enterotoxins (8). UG upregulation in zinc depletion correlated well with data from intervention trials that show a reduction in the incidence of diarrhea with zinc supplementation (9, 10). Initially identified as an EST, we subsequently cloned the rat UG gene using the differential display sequence information as the initial basis (11). UG expression was found primarily in the gastrointestinal tract with very little expression in the thymus and kidney and none in the other tissues examined. A polyclonal antibody developed from a 15–amino acid peptide with all three possible disulfide configurations of the active hormone was used for immunohistochemical and Western blotting studies (12).
UG peptides were localized primarily to enterochromaffin cells of the upper villus and were more abundant in intestines of zinc-deficient than zinc-adequate rats. Western blotting yielded comparable relative results. In situ hybridization of UG mRNA confirmed both the cell type and abundance that were demonstrated at the protein level. To our knowledge, this data set provides one of the first examples of successful analysis of differential expression at the transcriptome level and subsequent confirmation at the protein level. Comparable experiments showed that renal accumulation of UG was also elevated with zinc deficiency but was a consequence of increased intestinal synthesis and secretion of the prohormone (13).
Further analysis of the relationship of UG upregulation to the diarrhea that is observed in children, which is responsive to zinc supplementation, requires analysis of UG levels in appropriate subjects. Our experiments with rats do show a rapid reduction in UG expression upon zinc repletion, which agrees with the reduction in diarrhea that was observed in zinc-supplemented children (9,
10). Furthermore, interleukin-1
, which is a major factor in the induction of intestinal inflammation during infection with enteric pathogens (14), enhances the expression of both UG and inducible nitric oxide synthase to a greater extent in zinc-deficient rats than those fed adequate zinc (Cui et al., unpublished results). Expression of inducible nitric oxide synthase is also reduced upon zinc repletion. These findings are relevant because proinflammatory conditions must contribute to the morbidity that is observed in the intervention trials for diarrhea (9,
10).
The cDNA-array experiments that focus on the rat small intestine have provided information supplemental to that described above. These experiments used proprietary membrane arrays with 
1,200 cDNA segments for known rat genes (15). Data were analyzed using the significance analysis of microarrays (SAM) procedure (16) for comparison of three replicate arrays for each dietary treatment with subsequent confirmation by Q-PCR. Dysregulated genes identified by this profiling were from a variety of functional categories that include signal transduction, growth, transcription, redox and energy utilization. SAM uses a repeated-measures variance and mean differences, not fold changes, to assign significance. Initially, >60 genes were identified as upregulated and >60 genes were downregulated. SAM calculations lowered these to 25% of both lists. The fold changes were from +2.9 to -2.1 and are approximately what was reported for experiments with integrative systems rather than cultured cells. Frequently, mention is made in the array literature to ignore genes with fold changes of <2x, yet our experience has been that if they are statistically validated, lesser fold changes are relevant.
Collectively, these array experiments show that the confirmation step by Q-PCR is essential but can also include RNA from other treatment groups (e.g., food restricted) so that other factors influenced by a single micronutrient deficiency can be evaluated. These array experiments also point to the issue of detection sensitivity. A ZnT-1 cDNA was present on the array we used, yet phosphor imaging did not detect it as an expressed gene. Other experiments have demonstrated that ZnT-1 mRNA is expressed in small intestine at an abundance that is detectable by Northern analysis (17). This clearly indicates that sensitivity is a limitation of cDNA array analysis, and this limitation needs to be considered within the context of nutrition experiments aimed at functional outcomes.
Murine thymus. The impact of deficient zinc intake on immune function is well described (18). Failure of innate immunity, thymic atrophy and lymphopenia are among the features. Reasoning that changes in gene expression in the thymus gland might initiate such phenotypic events, we used a model of murine zinc deficiency with outbred mice to profile gene-expression changes. A 3-wk deficiency, before the thymocytes would show changes in the cell-surface markers CD3, CD4 or CD8 as measured by cell sorting, was used (19). Indices of deficiency and effects on gene expression included reduced pancreatic MT protein and reduced thymic MT mRNA. Thymic gene expression was profiled by both cDNA array and differential display as described above.
The murine arrays used were constructed with 1,200 cDNA segments of genes with established functions. Of these, 230 genes were found to be expressed in the thymus with hybridization intensities twofold over the background. The correlation coefficient was 0.99 for comparison of the intensities of expression between zinc-normal and -deficient mice, which indicates that the majority of the genes did not vary greatly due to withdrawal of zinc from the diet. These data (as shown for rat small intestine) further demonstrate that moderate zinc deficiency does not produce a global reduction in gene expression. Only four genes were differentially expressed >1.5-fold. These were subsequently confirmed by Q-PCR.
Relative to the zinc-adequate group, zinc deficiency produced changes for these genes: myeloid cell leukemia sequence-1 decreased 1.7-fold, DNA damage repair and recombination protein-23B (RAD23B) increased 1.8-fold, mouse laminin receptor increased 2.3-fold and lymphocyte-specific protein kinase (LCK) increased 1.5-fold. Mouse laminin receptor and myeloid cell leukemia sequence-1 are associated with preleukemic thymus and thymic carcinomas, respectively (20, 21). RAD23B interacts with ubiquitin and the regulatory S5a subunit domain of the 26S proteosome and thus impacts protein degradation. DNA damage induced by micronutrient deficiencies including zinc has been implicated in cancer initiation (22). Upregulation of the RAD23B gene may reflect an attempt by the thymus to reverse DNA damage.
The upregulation of LCK in these cDNA array studies was of particular interest. Cysteines of the cytoplasmic terminus of the CD4 receptor provide a zinc occupancy site with cysteines of the cytoplasmic LCK protein (23). Without zinc occupancy, these domains do not interact; hence there is no signal transduction to produce T-cell differentiation or activation (23, 24). A comparable scenario could occur in the zinc-deficient thymus. Previously, Lepage and co-workers (25) demonstrated that LCK protein levels increased in splenocytes during zinc deficiency. We were able to confirm LCK protein upregulation during zinc deficiency and expand this dysregulation to the thymus (19). Zinc deficiency causes a reduction in T-cell numbers and maturation. Therefore, it could be argued that LCK upregulates through a feedback signal in an attempt to correct T-cell developmental defects caused by the lack of Zn(II). Decreased thymic MT expression is observed in zinc deficiency. Because MT was proposed to serve as a Zn(II)-donor/acceptor (26) and has been proposed to be the zinc source for the LCK signal transduction pathway (24), a reduction in MT caused by zinc deficiency could influence signal transduction. Another key gene related to immune function that is upregulated by zinc deficiency is the T-cell cytokine receptor (TCCR). This newly described cytokine receptor aids in development of Th1-type (cell-mediated) immune responses (27). In contrast to LCK, this dysregulation was identified by differential display. Zinc deficiency has been shown to decrease the Th1 response with no change in the Th2 response (28, 29). An imbalance of Th1/Th2 would occur with upregulation of the TCCR gene. Our finding with TCCR could also be the manifestation of an attempt to correct, through upregulation, T-cell dysfunction related to dietary zinc deficiency.
A family of heat shock genes was found to be downregulated in deficiency. These are heat shock protein (Hsp) 40, Hsp 60, and heat shock cognate 70 (Hsc 70). Each of these proteins influences the folding of newly synthesized polypeptides by providing a chaperoned environment coupled to translation (30). Hsc 70 is an ATPase that binds hydrophobic proteins, whereas Hsp 40 stabilizes Hsc 70–substrate complexes by promoting ATP hydrolysis. Hsp 60 is a chaperonin (cylindrical protein complex) found in mitochondria that also participates in ATP-dependent protein folding. It could be argued that altered thymic cell activity produced by zinc deficiency would decrease protein folding and lead to a compensatory reduction in expression of genes required for folding. Alternatively, the decrease may reflect a limited ability during zinc deficiency to respond to stress-related denatured or improperly folded proteins, which are thought to be potentially toxic to cells.
Human mononuclear cells. We used THP-1 cells, which are a human monocytic leukemic cell line, as a cell-culture model to replicate expression changes observed in peripheral blood mononuclear cells that were purified from blood samples of human subjects. Routinely we use the cell-permeable zinc chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) to produce zinc deficiency in THP-1 cells (31). TPEN rapidly depletes cellular zinc concentrations in amounts of 5 and 10 µmol. Peripheral blood mononuclear cells obtained from volunteers exhibited similar levels of cellular zinc depletion with TPEN (31). For zinc supplementation of these cells, 80 µmol of zinc/L was used, which markedly increased the intracellular zinc concentrations. Zinpyr-1 was used as a fluorescent indicator to monitor intracellular zinc concentrations and distributions. MT and calreticulin (CRT) mRNA, both metal response element–regulated genes (32, 33), and Zip1 and Zip2 mRNA were used as indicative of changes expected of zinc-regulated genes in mononuclear cells. Zinc-depleted cells had reduced MT, CRT and Zip1 mRNA with increased Zip2 mRNA levels. Zinc-supplemented cells showed greater MT and CRT expression with slightly depressed Zip1 and Zip2 mRNA (31; Green et al., unpublished results).
For these experiments, cDNA arrays with human genes specifically applicable to hematology and immunology were used. Both zinc deficiency and supplementation produced changes of more than twofold for genes of high and low abundance. Of initial note was a decrease in ubiquitin and an increase in interferon regulatory factor 7 in zinc-deficient cells, whereas supplementation increased ubiquitin, interferon regulatory factor 5 and DNA topoisomerase expression. Presently, functional correlations with these findings are not possible and await more extensive cDNA array analyses, which are now in progress.
An emerging genotypic profile of zinc deficiency
Although cDNA arrays and to a lesser extent differential display are at the forefront of identification of those genes that are sensitive to changes in zinc nutrition, other methods such as protein-protein interaction studies, Western analyses, Q-PCR, sequence homology studies of open reading frames and promoter analysis have also been very fruitful. Proteomics has yet to be extensively used for studies on trace element nutrition. Nevertheless, the progress that has been made with tissues from zinc-deficient or zinc-supplemented rodents and from zinc-depleted and -supplemented human cells are among few demonstrations of transcriptome-level analysis that have been subsequently verified at the protein level.
Of particular note is the ability to place a number of genes within a functional context for consequences of inadequate zinc intake or the effects of zinc supplementation. Table 1 presents a short list of some genes that are found to be influenced by zinc nutrition. The majority of zinc-regulated genes are not those that code for proteins with enzymatic activity. In that context, it is not surprising that the first genes identified at both the transcriptome and protein levels are regulatory proteins. Upregulation of UG helps to explain the effect of zinc supplementation on the reduction of morbidity associated with diarrhea. Similarly, upregulation of LCK protein clearly relates to dysregulation of T-lymphocyte development and activation associated with zinc deficiency. Both of these findings could have occurred by chance, but considering that these effects have been confirmed at the mRNA level in addition to the protein level through Western analysis and, in the case of UG immunohistochemistry, reversal upon zinc repletion, a vision of the genetic profile of zinc deficiency may be emerging.
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Understanding the roles of individual transporters, the families of these transporters and transporters for multiple cations of nutritional interest provides an opportunity to understand zinc metabolism as never before. Experiments that have led to the identification of eukaryotic zinc transporters have used primarily mutagenized and/or transfected mammalian cells or yeast. For the purposes of this review, we focus on the ZnT family (ZnT-1 through ZnT-6) and the Zip family (Zip1 through Zip4). Identification and characterization of these transporter genes have resulted from the research of a number of laboratories (34– 45). Our interest in these transporters is to use this information to understand how their coordinated regulation by dietary intake levels and physiologic stimuli, e.g., hormones and cytokines, influences zinc metabolism.
We observed that expression of ZnT-1, ZnT-2 and ZnT-4 mRNA was tissue specific (46). These transporters also exhibited marked differences under steady-state conditions in regulation by dietary zinc; ZnT-2 was the most sensitive and ZnT-4 was the least sensitive. The kidney provides an excellent example of this response. ZnT-2 mRNA levels decrease 75% in zinc-deficient rats (<1 mg of zinc/kg for 15 d) and nearly double compared with controls (30 mg of zinc/kg) when high zinc (180 mg of zinc/kg) is fed (46). We routinely use renal ZnT-2 mRNA as one of the battery of zinc-responsive genes that are used for zinc-status assessment for experiments with rats (15). Kidney ZnT-1 mRNA responds similarly but the magnitude of change is less, whereas ZnT-4 mRNA does not change under these conditions.
ZnT expression also changes dramatically during gestation and lactation in maternal and fetal or pup tissues (Liuzzi et al., unpublished observations). For example, in maternal small intestine, ZnT-2 protein abundance as measured by Western analysis is high during gestation but is undetectable after day 1 of lactation. ZnT-1 remains constant, whereas ZnT-4 protein levels double during lactation. Our interpretation of these data is that ZnT-2 may serve a storage function for excess zinc. Such a reserve would be beneficial to the dam during pregnancy and the beginning of lactation. A mutation in the ZnT-4 gene was identified as being responsible for the murine lethal milk (lm) syndrome (35) and could explain the low zinc levels in milk produced by dams with the defective genotype. However, ZnT-4 is constitutively expressed in most tissues examined, which suggests that this transporter has a role in maintaining a uniform zinc supply to tissues. ZnT-1 may serve a protective function in some cell types and assist in coordinated zinc efflux and acquisition in other situations. In collaborative studies, ZnT-1 was demonstrated to reduce Zn(II) toxicity in neuronal (PC12) cells (47). In contrast, in mouse genotypes that are MT null or overexpress MT, intestinal and hepatic ZnT-1 expression is normal, although the ZnT-1 gene retains the ability to be markedly upregulated by zinc loading (48).
Some of the most informative data regarding the relationship of zinc transporters to their regulation and function comes from immunohistochemical studies of tissues. Much of the molecular genetics of transporters was carried out with transfected cells and fusion proteins without a clearly defined physiologic background. Although interesting, these approaches do not provide the systemic, cytokine and hormonal environments that are found in intact animals. Our experiments with ZnT-1 localization have focused on the intestine. This transporter was most highly expressed in duodenum and jejunum and was most abundant in villus rather than crypt cells. Immunofluorescence showed ZnT-1 localization to enterocytes along the entire villus (17). Of note was the lack of any ZnT-1 signal from cells of the lamina propria or goblet cells, which suggests a role in some aspect of zinc utilization or processing by enterocytes. Because ZnT-1 was most abundant near the basal lateral membrane of the small intestine of adult male rats and this transporter has a putative export function (34), we proposed a zinc-acquisition role for ZnT-1 in the intestine. Fluorescence appeared to be vesicular. Detection with chromagens and use of higher magnification of digital images support the vesicular location.
In the pregnant and lactating rat, intestinal ZnT-1 is distributed differently. At day 1 of lactation, ZnT-1 appears in vesicles oriented in a line that closely approximates the location of the trans-Golgi network. Later in lactation, ZnT-1 is found throughout enterocytes but still appears vesicular. ZnT-2 is oriented apically near the brush-border membrane, but localization follows the transient expression of this transporter, which is highest in late gestation/early lactation. ZnT-4 is distributed throughout enterocytes and also shows a vesicular localization. In rat placenta, both ZnT-1 and ZnT-4 are localized to the visceral splanchnopleure of the villus yolk sac. This suggests a role for both proteins in zinc transport to the fetus. ZnT-1 and ZnT-4 in neonatal rat small intestine show a developmental regulation with both proteins localized in similar fashion to those of the dam.
We have also used THP-1 cells to examine ZnT and Zip-transporter expression after zinc-depletion treatment with TPEN. From Q-PCR data, which is an analytical technique that generates a quantity, we estimate a crude relative abundance of these transporter mRNA (compared with MT mRNA) as follows:
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when the culture medium was 3 µmol of zinc/L (31). As a function of length of zinc depletion, there is a marked reduction in expression of each gene except for Zip2, which increases as shown in
Figure 1. ZnT-1 is the most rapidly changed and does so before MT, the prototype zinc-regulated gene. One could reason from these data that expression of the ZnT genes is decreasing because zinc sequestration by cells is not needed. This follows the efflux function proposed for these transporters. However, if efflux was related to expansion of vesicular zinc concentrations, it could be argued that ZnT expression should increase during zinc depletion as a way to conserve zinc. In contrast, Zip expression would be expected to increase, to produce an increase in Zn(II) influx and attempt to rescue the cells from zinc depletion. Zip2 follows that mode, but Zip1 does not. This difference raises questions about the function of the specific Zip transporters and provides interesting research opportunities to examine mechanisms of transporter gene transcriptional regulation.
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FOOTNOTES |
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2 Research reviewed in this paper was supported by the National Institute of Diabetes and Digestive and Kidney Diseases and the Boston Family Endowment Funds of the University of Florida.
4 Present address: Instituto de Biologia Experimental, Universidad Central de Venezuela, 1041-A Caracas, Venezuela.
5 Present address: Department of Medicine, University of California, San Francisco, CA 94121.
6 Abbreviations used: CRT, calreticulin; EST, expressed sequence tag; Hsc 70, heat shock cognate 70; Hsp, heat shock protein; LCK, lymphocyte-specific protein tyrosine kinase; MT, metallothionein; Q-PCR, quantitative real-time polymerase chain reaction; RAD23B, DNA damage repair and recombination protein-23B; SAM, significance analysis of microarrays; TCCR, T-cell cytokine receptor; TPEN, N,N,N'N'-tetrakis(2-pyridylmethyl)ethylenediamine; UG, uroguanylin; ZnT, zinc transporter.
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LITERATURE CITED |
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TOPABSTRACTLITERATURE CITED |
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