Available online at www.annclinlabsci.org 230 Annals of Clinical & Laboratory Science, vol. 35, no. 3, 2005 Overview: Global Genomic Approaches to the Iron-Regulated Proteome Ying Liu, Zvezdana Popovich, and Douglas M. Templeton Department of Laboratory Medicine and Pathobiology, University of Toronto,Toronto, Canada Abstract. Iron interacts with cells to regulate the proteome through complex effects on gene expression. In simple organisms such as bacteria and yeast, intra- and extra-cellular iron influences gene expression through defined signal transduction pathways. In higher organisms, effects are probably mediated at the transcriptional level through secondary effects of reactive oxygen species, while post-transcriptional effects operate through well-defined pathways involving iron-regulatory proteins. To investigate the impact of iron levels on gene expression and the proteome, approaches such as differential display and subtractive hybridization have the advantage of surveying the entire geneome. However, they are technically demanding and have given way to microarray techniques. To date, numerous microarray experiments with various organisms have not yielded any definitive picture of the role of iron. Common themes throughout such studies are that both iron excess and iron depletion influence expression of proteins related to energy metabolism, cell proliferation, matrix structure, and the metabolism of iron itself. That no consistent set of genes is involved from one study to the next probably results both from the uncertainties inherent in the technique and the biological variability of the systems under study. We briefly describe two types of iron-dependent microarray experiments from our laboratory to examine major cellular targets of iron toxicity. Using Affymetrix oligonucleotide arrays with cardiac cells, we found several hundred genes whose mRNA levels were affected by iron, including an increase in several genes responding to oxidative stress and a decrease in several kinases and phosphatases. In a simpler experiment using a human liver cell line with a limited cDNA array, we targeted 13 genes affected by iron chelation. Metabolic pathway analysis shows links of 5 of these through phorbol ester responsiveness, and additional links through prostaglandin E2. We conclude that definitive understanding of the complex iron-regulated proteome requires global gene approaches and rigorous interlaboratory standardization. (received 22 April 2005; accepted 28 April 2005) Keywords: gene expression, microarray, genome, iron overload, iron chelation, iron-responsive elements Introduction synthesis to maintain the blood’s oxygen-carrying capacity. On the other hand, iron’s redox properties Iron is required by living organisms for a variety of contribute to its toxicity. Through participation in purposes related to its favourable redox properties Fenton chemistry, iron produces reactive oxygen and rich coordination chemistry. Thus, in the species (ROS) that are harmful to biological human organism, it is essential for the mitochondrial molecules. And replete iron stores also increase our electron transport chain, as part of cytochromes and susceptibility to infection; many opportunistic iron-sulfur proteins, and for nucleic acid synthesis organisms rely on the host to supply precious iron. as a cofactor in ribonucleotide reductase. The body As might be expected of such a bioactive metal, iron utilizes about 20 mg of Fe per day for hemoglobin also influences cellular responses and phenotype through diverse effects on gene expression. Our Address correspondence to Douglas M. Templeton M.D., purpose here is to survey various studies that have Ph.D., Department of Laboratory Medicine and Pathobiology, taken a broad genome/proteome-based approach to Medical Sciences Building Rm 6302, University of Toronto, 1 understanding the role of iron in orchestrating King’s College Circle, Toronto M5S 1A8, Canada; tel 416 978 3972; fax 416 978 5959; e-mail [email protected]. cellular biochemistry. 0091-7370/05/0300-0230. $2.50 © 2005 by the Association of Clinical Scientists, Inc. 02 Liu 230-239 230 7/20/05, 1:11 PM Genomic approaches to iron regulation 231 Major steps in understanding iron metabolism TonB in E. coli. In situations of low iron, the Fec were made over the last decade [1]. A specific role proteins are transcribed following removal of Fur for iron in regulating genes involved in its own repression, but the transport system is inactive. metabolism and trafficking at a post-transcriptional When a low concentration of ferric dicitrate is level [2-4] became textbook examples of such a mode present in the cell’s vicinity, it is sensed by binding of regulation. A perhaps reawakened interest in the to FecA in the outer membrane. This leads to metal saw classical molecular biological approaches interaction of FecA with a protein complex that such as differential display and subtraction cloning includes FecR and TonB on the inner membrane, used to address more global effects of iron [5,6]. and this induces a conformational change in FecR. Recently, microarray technology has dominated such FecR then interacts with FecI in the cell, activating efforts. But iron remains somewhat enigmatic. it to bind to the FecA promoter, recruit RNA Multiple effects on gene expression have been polymerase, and drive transcription of genes of the proposed but remain hard to categorize in terms of Fec iron transport system (FecA, FecB, FecC, etc.) a unified response [7]. And, in view of its potential that are contiguously arranged on the chromosome. effects on multiple signaling mechanisms, iron This leads to increased iron uptake through FecA, overload, in cultured cells at least, often has which is thus both a regulator and a transporter. surprisingly little effect. Distinguishing acute toxic Again, this system is repressed by Fur at high iron responses from adaptive ones is a goal of such studies. concentrations. A number of other bacteria express The lack of a dramatic effect of iron overload components homologous to those of the Fec and on gene expression notwithstanding, iron does Fur systems, and signal transduction appears to be influence a variety of cellular processes. We have a general theme in iron-dependent bacterial gene recently reviewed effects that go beyond regulation regulation. Another good example is the PmrA/ of genes related to iron metabolism itself (operating PmrB system of Salmonella that responds to extra- at both transcriptional and translational levels) to cellular Fe3+ to activate a regulon involved in iron genes grouped as affecting oxidative stress responses resistance [9]. (eg, glutathione peroxidase, heme oxygenase-1, metallothionein); tissue fibrosis (eg, collagen, TGF- Transcriptional and Post-transcriptional Regulation β); energetics of metabolism (eg, aldolase, lactate dehydrogenase); and cell cycle control (eg, retino- In general, transcriptional regulation by iron in blastoma protein, p21, various cyclins) [7]. higher animals is poorly understood. Generation of ROS by iron may be a general means of gene Signaling Evoked by Iron regulation through the ROS-activated transcription factor NF-κB [10,11]. For instance, an NF-κB Studies with bacteria have linked iron with signal binding site is found in the ferritin H chain gene transduction mechanisms that terminate in promoter region [12]. ROS also lead to lipid per- regulating gene expression, and these may prove to oxidation. Products of lipid peroxidation in turn be instructive for understanding iron in higher activate transcription factors Sp1 and Sp3, account- organisms. The E. coli Fur protein has homologues ing at least in part for an increased expression of in many bacteria. It can act directly as a transcript- a1(I) collagen [13,14]. On the other hand, hypoxia- ional repressor when Fe2+ is present as a cofactor, like responses play a central role in gene regulation and turns off genes related to iron uptake. When by iron chelators [7]. Hypoxia-inducible factor iron is scarce, then, derepression alone can increase (HIF-1α) is a transcription factor that binds to a synthesis of iron transport proteins and enzymes of hypoxia-responsive element (HRE) in a number of siderophore biosynthesis [8]. However, in other target genes. The iron chelator deferoxamine (DFO) systems, positive regulation is required for synthesis mimics the effects of hypoxia on a number of genes of the iron transport systems. A case in point is [15,16] including erythropoietin [17] and the ferric citrate signaling through the Fec proteins and transferrin receptor [18]. A possible mechanism 02 Liu 230-239 231 7/20/05, 1:11 PM 232 Annals of Clinical & Laboratory Science, vol. 35, no. 3, 2005 involves the Fe(II)/2-oxoglutarate-dependent dioxy- structure with a base-paired stem of about 10 base genase, which hydroxylates critical proline and pairs in length, a central unpaired loop of sequence arginine residues in HIF-1α under normoxic and CAGUG, and a conserved C, 5 bases upstream of iron-replete conditions, targeting HIF-1α for this loop, that forms an unpaired bulge in the stem degradation by the proteasome [19,20]. and is necessary for protein binding. IREs in the 5' Post-transcriptional regulation in eukaryotes of UTR of transcripts occur in single copies, and are several genes controlling iron metabolism and energy found in ferritin, ferroportin, m-aconitase, succinate utilization is better understood. It involves iron- dehydrogenase, and δ−ALAS mRNAs. Their regulatory elements