Raymond K. Blanchard

A global view of the selectivity of zinc deprivation and excess on genes expressed in human THP-1 mononuclear cells

Among the micronutrients required by humans, zinc has particularly divergent modes of action. cDNA microarray and quantitative PCR technologies were used to investigate the zinc responsiveness of known genes that influence zinc homeostasis and to identify, through global screening, genes that may relate to phenotypic outcomes of altered dietary zinc intake. Human monocytic/macrophage THP-1 cells were either acutely zinc depleted, using a cell-permeable zinc-specific chelator, or were supplemented with zinc to alter intracellular zinc concentrations. Initially, genes associated with zinc homeostasis were evaluated by quantitative PCR to establish ranges for fold changes in transcript abundance that might be expected with global screening. Zinc transporter-1 and zinc transporter-7 expression increased when cellular zinc increased, whereas Zip-2 expression, the most zinc-responsive gene examined, was markedly increased by zinc depletion. Microarrays composed of ≈22,000 elements were used to identify those genes responsive to either zinc depletion, zinc supplementation, or both conditions. Hierarchal clustering and ANOVA revealed that ≈5% or 1,045 genes were zinc responsive. Further sorting based on this pattern of the zinc responsiveness of these genes into seven groups revealed that 104 genes were linearly zinc responsive in a positive mode (i.e., increased expression as cellular zinc increases) and 86 genes that were linearly zinc responsive in a negative mode (i.e., decreased expression as cellular zinc increases). Expression of some genes was responsive to only zinc depletion or supplementation. Categorization by function revealed numerous genes needed for host defense were among those identified as zinc responsive, including cytokine receptors and genes associated with amplification of the Th1 immune response.

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.

Regulation of Zinc Metabolism and Genomic Outcomes

Differential mRNA display and cDNA array analysis have identified zinc-regulated genes in small intestine, thymus and monocytes. The vast majority of the transcriptome is not influenced by dietary zinc intake, high or low. Of the genes that are zinc regulated, most are involved in signal transduction (particularly influencing the immune response), responses to stress and redox, growth and energy utilization. Among the genes identified are uroguanylin (UG), cholecystokinin, lymphocyte-specific protein tyrosine kinase (LCK), T-cell cytokine receptor, heat shock proteins and the DNA damage repair and recombination protein-23B. Zinc transporters (ZnT) help regulate the supply of this micronutrient to maintain cellular functions. Expression of ZnT-1 and -2 is regulated by dietary zinc in many organs including small intestine and kidney. ZnT-4 is ubiquitously expressed but is refractory to zinc intake. Expression of ZnT-1, -2 and -4 changes markedly during gestation and lactation from highly abundant to undetectable. Each ZnT has an endosomal-like appearance in the tissues examined. Upregulation of ZnT-1 and ZnT-2 by dietary zinc strongly implicates these transporters in zinc acquisition and/or storage for subsequent systemic needs. THP-1 cells were used as a model to examine the response of human cells to changes in zinc status. Based on mRNA quantities, Zip1 and ZnT-5 were the most highly expressed. Zinc depletion of these cells decreased expression of all transporters except Zip2, where expression increased markedly. Collectively, these findings provide a genomic footprint upon which to address the biological and clinical significance of zinc and new avenues for status assessment.

Dietary zinc modulates gene expression in murine thymus: Results from a comprehensive differential display screening

Differential mRNA display was used to comprehensively screen the murine thymic transcriptome for genes modulated in vivo by dietary zinc. A moderate feeding protocol rendered young adult, outbred mice zinc-deficient and zinc-supplemented without alterations in feeding behavior or growth. However, these levels of deficiency and supplementation altered specific mRNA abundances in a manner detectable by differential display. In total, 240 primer-pair combinations were used to generate >48,000 interpretable cDNA bands derived from thymic total RNA, of which only 265 or 0.55% were identified as zinc-modulated under these moderate dietary conditions. The most strongly zinc-modulated cDNAs identified by display were reamplified and sequenced. No cDNAs encoding zinc-metalloenzymes or zinc-finger transcription factors were identified as zinc-modulated in this global screening. Those zinc-regulated genes independently confirmed by quantitative PCR included: heat shock proteins 40 and 60; heat shock cognate 70; histocompatibility 2, class II antigen A, α; and the T cell cytokine receptor. In addition, a variety of transcription- and translation-related factors (such as ribosomal proteins L3, L5, and L28; nuclear matrix protein 84; matrin cyclophilin; the H3 histone family 3A protein; β2 microglobulin; and a cleavage and polyadenylation factor) were identified as zinc-modulated. These profiling data show that differential expression of genes in the thymus in response to the dietary zinc supply precedes many of the phenotypic effects on thymic function associated with severe zinc restriction or supplementation. Several genes involved in T cell development were identified as regulated by zinc and will be targets to evaluate the effects of zinc on immune function.

Regulation of the ferredoxin component of renal hydroxylases at transcriptional and postranslational levels and of the protein inhibitor of cyclic AMP-dependent kinase.

We have studied two proteins potentially involved in the regulation of the 25-OH-D-1-hydroxylase, which is located in the renal mitochondria and which is responsible for the production of the steroid hormone 1,25(OH)2D3. The endogenous inhibitor of cyclic AMP-dependent protein kinase, PKI, is down regulated by 1,25(OH)2D3. Having cloned and sequenced PKI cDNA, we studied its message levels and found them to be regulated by 1,25(OH)2D3 tissue specifically in the kidney and in kidney cell culture. In other experiments we over expressed the ferredoxin component of the 1-hydroxylase and found it to be physically and chemically indistinguishable from those of classic steroidogenic tissues. The mRNA encoding the ferredoxin component is up-regulated by chronic vitamin D deficiency, which at the same time leads to sustained elevation in 1-hydroxylase activity; no short term effect of 1,25(OH)2D3 on ferredoxin mRNA in kidney cell culture could be demonstrated. Finally, there was an association between decreased phosphorylation of ferredoxin and decreased 1-hydroxylase activity brought about by treatment of cultured kidney cells with TPA. Control of the renal signaling events involved in the production of 1,25(OH)2D3 remains a fruitful area of investigation in the field of the metabolism and actions of vitamin D and its metabolites.