cDNA array analysis identifies thymic LCK as upregulated in moderate murine zinc deficiency before T-lymphocyte population changes.

The detrimental sequelae of severe zinc deficiency on the thymus and T-lymphocyte compartment of the mammalian immune system have been established, but underlying mechanisms remain unknown. Hypothesizing that the alterations in T-lymphocyte number and function observed during zinc deficiency may result from changes in gene expression, we sought to compare thymic mRNA expression profiles of zinc-deficient and zinc-normal mice utilizing cDNA arrays. For our murine model described herein, 3 wk of dietary zinc deficiency did not perturb food intake or growth rate in young adult, outbred mice, but significantly depressed multiple parameters of zinc status. Furthermore, fluorescence-activated cell sorting (FACS) analysis demonstrated no changes in thymocyte populations expressing the cell surface markers CD3, CD4 or CD8, establishing that observed changes in mRNA abundances were not attributable to different thymocyte populations. Yet notably, at this moderate level of zinc deficiency, cDNA array analysis identified four potentially zinc-regulated mRNAs whose modulation was confirmed independently, twice, using both semiquantitative and real-time quantitative reverse transcription-polymerase chain reaction. Expression of one of these genes (myeloid cell leukemia sequence-1) was depressed, whereas the others [DNA damage repair and recombination protein 23B, the mouse laminin receptor and the lymphocyte-specific protein tyrosine kinase (LCK)] were elevated in the zinc-deficient mice. Further Western analysis demonstrated that the zinc binding protein LCK was elevated in these zinc-deficient mice. Results demonstrate that 3 wk of dietary zinc insufficiency can alter specific thymic mRNA and protein abundances before alterations occur in thymocyte development as detectable by FACS analysis.

Modulation of intestinal gene expression by dietary zinc status: effectiveness of cDNA arrays for expression profiling of a single nutrient deficiency.

Mammalian nutritional status affects the homeostatic balance of multiple physiological processes and their associated gene expression. Although DNA array analysis can monitor large numbers of genes, there are no reports of expression profiling of a micronutrient deficiency in an intact animal system. In this report, we have tested the feasibility of using cDNA arrays to compare the global changes in expression of genes of known function that occur in the early stages of rodent zinc deficiency. The gene-modulating effects of this deficiency were demonstrated by real-time quantitative PCR measurements of altered mRNA levels for metallothionein 1, zinc transporter 2, and uroguanylin, all of which have been previously documented as zinc-regulated genes. As a result of the low level of inherent noise within this model system and application of a recently reported statistical tool for statistical analysis of microarrays [Tusher, V.G., Tibshirani, R. & Chu, G. (2001) Proc. Natl. Acad. Sci. USA 98, 5116-5121], we demonstrate the ability to reproducibly identify the modest changes in mRNA abundance produced by this single micronutrient deficiency. Among the genes identified by this array profile are intestinal genes that influence signaling pathways, growth, transcription, redox, and energy utilization. Additionally, the influence of dietary zinc supply on the expression of some of these genes was confirmed by real-time quantitative PCR. Overall, these data support the effectiveness of cDNA array expression profiling to investigate the pleiotropic effects of specific nutrients and may provide an approach to establishing markers for assessment of nutritional status.

Dietary zinc deficiency increases uroguanylin accumulation in rat kidney.

BACKGROUND: Zinc deficiency in humans produces a secretory diarrhea that is corrected by zinc supplementation. In rats, differential mRNA display analysis has shown that intestinal uroguanylin gene expression is increased in zinc deficiency. An endocrine axis involving intestinal uroguanylin and the kidney may exist. Therefore, we conducted this study to examine whether zinc deficiency would affect uroguanylin expression in the kidney of rats. METHODS: A purified diet, deficient or adequate in zinc content, was fed to rats. Preprouroguanylin mRNA was localized in kidney by in situ hybridization, and prouroguanylin/uroguanylin peptides were localized in the kidney by immunohistochemistry. Abundance was measured by Western blotting and slot blotting analyses. RESULTS: In situ hybridization demonstrated that preprouroguanylin mRNA-expressing cells were localized in the proximal tubules, being primarily limited to the cortical-medullary junction. Zinc deficiency did not alter the abundance or distribution of the mRNA. Immunohistochemistry, using a uroguanylin peptide-specific, affinity-purified antibody, demonstrated that immunoreactive uroguanylin peptide was localized to the same cells but that the staining was stronger in zinc-deficient rats. Western blotting analysis of kidney extracts showed that there was no difference in abundance of prouroguanylin between zinc adequate and deficient rats. However, slot blotting analysis demonstrated that the abundance of a low molecular weight immunoreactive peptide, presumably uroguanylin, was higher in extracts of zinc-deficient rats. CONCLUSION: The results suggest that production of prouroguanylin by the kidney, in contrast to the intestine, is not influenced by dietary zinc intake, but that higher amounts of uroguanylin in kidney extracts may reflect renal processing of the hormone obtained from the systemic circulation.

Differential regulation of zinc transporter 1, 2, and 4 mRNA expression by dietary zinc in rats.

Zinc metabolism is well regulated over a wide range of dietary intakes to help maintain cellular zinc-dependent functions. Expression of transporter molecules, which influence zinc influx and efflux across the plasma and intracellular membranes, contributes to this regulation. We have examined in rats the comparative response of zinc transporters 1, 2, and 4 (ZnT-1, ZnT-2 and ZnT-4) to dietary zinc. ZnT-1 and ZnT-4 are expressed ubiquitously, whereas ZnT-2 is limited to small intestine, kidney, placenta and, in some cases, the liver. When zinc intake was low (<1 mg Zn/kg), ZnT-2 mRNA was extremely low in small intestine and kidney compared with an adequate intake (30 mg Zn/kg). ZnT-1 and ZnT-2 mRNAs were markedly greater in both tissues when a supplemental zinc intake (180 mg Zn/kg) was provided. ZnT-4 was refractory to changes in zinc intake. When zinc was provided as a single oral dose (70 mg/kg body), ZnT-1 and ZnT-2 mRNA levels were increased many fold in small intestine, liver and kidney, whereas ZnT-4 gene expression was not changed. The expression of ZnT-1 and ZnT-2 is comparable to zinc-induced changes in metallothionein mRNA levels, suggesting a similar mode of regulation for these genes. The relative differential in regulation by zinc is ZnT-2 > ZnT-1 > ZnT-4. These data provide evidence that, in an animal model, zinc transporter expression is responsive to zinc under physiologically relevant conditions.

Prouroguanylin overproduction and localization in the intestine of zinc-deficient rats.

Identification of the upregulation of preprouroguanylin mRNA in the rat small intestine during zinc deficiency provides a potential mechanistic link between production of the intestinal hormone uroguanylin and the diarrhea that may accompany zinc deficiency. In the current study, in situ hybridization demonstrated that the number of preprouroguanylin mRNA-expressing cells was significantly higher in zinc-deficient rats than in zinc-adequate rats. Immunohistochemical studies, with a uroguanylin peptide affinity-purified antibody, demonstrated that immunoreactivity was localized to the tips of villi of the duodenum and jejunum in zinc-adequate rats. However, positive cells were scattered throughout the villus of zinc-deficient rats. A subset of cells, perhaps enterochromaffin cells, exhibited the predominant staining, whereas no specific staining was found in goblet cells or lymphocytes of the lamina propria. Western blotting demonstrated that the expression of prouroguanylin in both duodenum and jejunum was elevated by dietary zinc depletion. These results show that dietary zinc deficiency upregulates prouroguanylin in intestinal cells, which is consistent with a role for uroguanylin in the etiology of diarrhea observed in human zinc deficiency.

Regulation of intestinal gene expression by dietary zinc: induction of uroguanylin mRNA by zinc deficiency.

The regulation of gene expression by nutrients plays an important role in the overall manifestations of nutritional deficiencies. Insufficient intakes of dietary micronutrients, such as zinc, produce profound effects in multiple organs and tissues. One of the major challenges, however, is to identify genes affected by changes in nutritional status. Differential display of mRNA has proved to be a valuable technique in meeting this challenge. In our ongoing search for genes responsive to dietary zinc, we compared small intestinal mRNA from rats that were fed zinc-deficient or -adequate diets using differential display to generate 3' anchored expressed sequence tags (EST). EST for intestinal mRNAs with altered expression due to zinc deficiency include two peptide hormones, intestinal fatty acid binding protein, intestinal alkaline phosphatase II, a proteasomal ATPase, cis-Golgi p28 and two subunits of the ubiquinone oxidoreductase. The EST for one of the hormones yielded the sequence for the 3' end of an mRNA encoding preprouroguanylin and was used to clone the remaining portion of the rat cDNA via 5' rapid amplification of cDNA ends. Northern blot analysis of RNA from rat intestine demonstrated that preprouroguanylin mRNA was 2.5-fold more abundant during zinc deficiency. Uroguanylin, a natriuretic peptide hormone, is an endogenous ligand for the same guanylate cyclase C that the Escherichia coli heat-stable enterotoxin (STa) binds when it causes secretory diarrhea by activating the cystic fibrosis transmembrane conductance regulator, thus altering fluid balance in the intestine. This suggests a mechanism whereby zinc deficiency could induce uroguanylin levels in the intestine and cause or potentiate diarrhea.

Structural characterization of the rat cysteine-rich intestinal protein gene and overexpression of this LIM-only protein in transgenic mice.

Cysteine-rich intestinal protein (CRIP) has a double zinc-finger motif called the LIM domain. The most elementary member of the Group 2 LIM-only protein family, CRIP was initially identified as a developmentally regulated intestinal gene. Subsequently, it was found to be highly expressed in immune cells. The structural portion of the rat CRIP gene is comprised of five exons extending over i.8 kb, with the two zinc-finger motifs of the LIM domain being divided among the first three exons. In addition to transcriptional regulatory elements previously identified in the promoter, consensus sequences for AP-1, AP2, Sp-1, and a glucocorticoid response element are located within the first intron. We have developed a line of transgenic mice that overexpress the rat CRIP gene with an expression profile that mirrors that of the endogenous gene. Driven by the homologous rat CRIP promoter, expression increased threefold to sevenfold in intestine, thymus, spleen, and lung over endogenous levels. The transgenic mice had only about 50% of the white blood cell count found in nontransgenic animals. Differential leukocyte counts showed transgenic animals had proportionately fewer lymphocytes and more monocytes, eosinophils, neutrophils. Flow cytometry data suggested that mice overexpressing CRIP have more CD4+/CD8+ thymic lymphocytes. These data suggest that CRIP plays a significant role in differentiation or maturation of cells with rapid turnover such as those found in the intestine and immune system.

Upregulation of rat intestinal uroguanylin mRNA by dietary zinc restriction.

A cDNA for the rat uroguanylin precursor was identified, by differential display of intestinal mRNA, as upregulated in zinc-deficient rats and subsequently was cloned. The cDNA and deduced amino acid sequences show a high degree of homology to human and opossum preprouroguanylin sequences. When used as a probe for Northern blot analysis of RNA from rat intestinal mucosa, the uroguanylin cDNA hybridized to a single species of mRNA that was 2.5-fold more abundant in zinc deficiency. A tissue distribution survey indicates that although the small intestine expresses a disproportionately high level of preprouroguanylin, this hormone precursor is also expressed in the colon, stomach, kidney, thymus, and testis. The induction by zinc deficiency is the first reported case of gene regulation for this hormone. These results also suggest a potential mechanism to explain, at least in part, the beneficial effects of zinc supplementation for secretory diarrhea prevalent in many areas of the world.

Human cysteine-rich intestinal protein: cDNA cloning and expression of recombinant protein and identification in human peripheral blood mononuclear cells.

Cysteine-rich intestinal protein (CRIP) is a small, 8.5-kDa protein with one double zinc-finger motif called a LIM domain. It is very abundant in intestine and some immune cells in rodents, and expression is influenced by development and the immune response. We have cloned a human CRIP cDNA from human small intestine poly(A)+ RNA by RT-PCR. Through sequencing, we found that the human intestinal CRIP protein (hCRIP) differed from the previously cloned rat CRIP by two amino acids (residues 8 and 58). hCRIP was expressed with the pET vector/bacterial system and isolated by gel filtration and ion-exchange chromatography. The protein was purified to homogeneity as confirmed by PAGE, Western blotting, and immunodetection. Recombinant hCRIP has a molecular mass of 8390 Da based on mass spectrum analysis. Southern analysis suggests that there are three copies of the CRIP gene in the human genome. hCRIP mRNA was detected by RT-PCR in human monocytes purified from peripheral blood and THP-1 cells, a human monocytic cell line. Incubation of THP-1 cells with 65Zn and chromatography of the cytosol show that a significant amount of the radioactivity is associated with CRIP as was shown previously for rat intestine. The results are consistent with a functional role for CRIP in proliferation/differentiation of specific cell types, particularly those associated with host defense.

Differential display of intestinal mRNAs regulated by dietary zinc.

Regulation of gene expression by zinc is well established, especially through the metal response elements of the metallothionein genes; however, most other aspects of the functions of zinc in gene expression remain unknown. We have looked for intestinal mRNAs that are regulated by dietary zinc status. Using the reverse transcriptase-PCR method of mRNA differential display, we compared intestinal mRNA from rats that were maintained for 18 days in one of three dietary groups: zinc-deficient, zinc-adequate, and pair-fed zinc-adequate. At the end of this period, total RNA was prepared from the intestine and analyzed by mRNA differential display. Under these conditions, only differentially displayed cDNA bands that varied in the zinc-deficient group, relative to the zinc-adequate groups, were selected. Utilizing two anchored oligo-dT3' PCR primers and a total of 27 arbitrary decamers as 5' PCR primers, our results yielded 47 differentially displayed cDNA bands from intestinal RNA. Thirty were increased in zinc deficiency, and 17 were decreased. Nineteen bands were subcloned and sequenced. Eleven of these were detectable on Northern blots, of which four were confirmed as regulated. Three of these have homology to known genes: cholecystokinin, uroguanylin, and ubiquinone oxidoreductase. The fourth is a novel sequence as it has no significant homology in GenBank. The remainder of those cloned included novel sequences, as well as matches to reported expressed sequence tags, and functionally identified genes. Further characterization of the regulated sequences identified here will show whether they are primary or secondary effects of zinc deficiency.

Apolipoprotein B mRNA editing is preserved in the intestine and liver of zinc-deficient rats.

Apolipoprotein B (apo B) mRNA editing is a site-specific, post transcriptional cytidine deamination reaction that generates apo B48 in the mammalian small intestine and in the liver of certain animals. This reaction is mediated by an enzyme complex that includes the catalytic subunit apobec-1, a zinc-dependent cytidine deaminase. To determine the importance of zinc status to apo B mRNA editing in vivo, we examined the effects of experimentally induced zinc deficiency in rats upon hepatic and serum lipid levels and several indices of apo B gene expression. Rats were either given unlimited access to or were pair-fed a semipurified zinc-supplemented (30 mg Zn/kg) diet or were fed a zinc-deficient diet (approximately 1 mg Zn/kg) for 17 d. Significant differences were detected in the ratio of serum apo B100/B48 in the unlimited access, zinc-supplemented group compared with either zinc-deficient rats or pair-fed controls. There were no alterations in hepatic triglyceride and cholesterol concentrations, hepatic apo B mRNA abundance or apo B mRNA editing in either the small intestine or liver. Taken together, these data suggest that the altered ratios of serum apo B isomorphs seen in zinc deficiency are not mediated through changes in hepatic or intestinal apo B mRNA editing.