From bloodjournal.hematologylibrary.org by guest on June 3, 2013. For personal use only. Prepublished online September 26, 2002; doi:10.1182/blood-2002-07-2140 Relationships and distinctions in iron regulatory networks responding to interrelated signals Martina Muckenthaler, Alexandra Richter, Niki Gunkel, Dieter Riedel, Maria Polycarpou-Schwarz, Sabine Hentze, Mechthild Falkenhahn, Wolfgang Stremmel, Wilhelm Ansorge and Matthias W Hentze Articles on similar topics can be found in the following Blood collections Gene Expression (1086 articles) Genomics (149 articles) Red Cells (1174 articles) Signal Transduction (1930 articles) Information about reproducing this article in parts or in its entirety may be found online at: http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://bloodjournal.hematologylibrary.org/site/subscriptions/index.xhtml Advance online articles have been peer reviewed and accepted for publication but have not yet appeared in the paper journal (edited, typeset versions may be posted when available prior to final publication). Advance online articles are citable and establish publication priority; they are indexed by PubMed from initial publication. Citations to Advance online articles must include the digital object identifier (DOIs) and date of initial publication. Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. Copyright 2011 by The American Society of Hematology; all rights reserved. From bloodjournal.hematologylibrary.org by guest on June 3,26, 2013. For personal use only. Blood First Edition Paper, prepublished online September 2002; DOI 10.1182/blood-2002-07-2140 Relationships and distinctions in iron regulatory networks responding to interrelated signals Martina Muckenthaler1, Alexandra Richter1, Niki Gunkel2, Dieter Riedel3, Maria Polycarpou-Schwarz1, Sabine Hentze1, Mechthild Falkenhahn4, Wolfgang Stremmel3, Wilhelm Ansorge1 and Matthias W. Hentze1* Running title: " IronChip" analysis of cellular iron metabolism Scientific Heading: Red cells Funds from the Gottfried Wilhelm Leibniz Prize to MWH were used to establish the "IronChip". We thank the Resource Center and Primary Database (RZPD) for the supply of IMAGE clones. Word count: 230 (abstract); 4831 (text) Keywords: iron metabolism, HFE, gene expression profiling, microarray, IronChip *Corresponding author 1 European Molecular Biology Laboratory, Meyerhofstrasse 1 D-69117 Heidelberg Germany Tel.: +49-6221-387501; FAX: +49-6221-387518 E-mail: Hentze@EMBL-Heidelberg.de 2 Intervet International, Germany 3 Department of Medicine, University of Heidelberg, Germany 4 Department of Biocomputing, Krebsforschungszentrum, Heidelberg, Germany 1 Copyright (c) 2002 American Society of Hematology From bloodjournal.hematologylibrary.org by guest on June 3, 2013. For personal use only. Abstract Specialized cDNA-based microarrays were developed to investigate complex physiological gene regulatory patterns in iron metabolism. Approximately 115 human cDNAs were strategically selected to represent genes involved either in iron metabolism or in interlinked pathways (e.g. oxidative stress, NO metabolism or copper metabolism), and immobilized on glass slides. HeLa cells were treated with iron donors or iron chelators, or were subjected to oxidative stress (H2O2) or NO (sodiumnitroprusside). In addition, we generated a stable transgenic HeLa cell line expressing the HFE gene under an inducible promoter. Gene response patterns were recorded for all of these interrelated experimental stimuli, and analyzed for common and distinct responses that define signalspecific regulatory patterns. The resulting regulatory patterns reveal and define degrees of relationship between distinct signals. Remarkably, the gene responses elicited by the altered expression of the hemochromatosis protein HFE and by pharmacological iron chelation exhibit the highest degree of relatedness, both for IRP- and non-IRP target genes. This finding suggests that HFE expression directly affects the intracellular chelatable iron pool in the transgenic cell line. Furthermore, cells treated with the iron donors hemin or ferric ammonium citrate display response patterns that permit the identification of the iron loaded state in both cases, and to discriminate between the sources of iron loading. These findings also demonstrate the broad utility of gene expression profiling with the “IronChip” to study iron metabolism and related human diseases. 2 From bloodjournal.hematologylibrary.org by guest on June 3, 2013. For personal use only. Introduction Iron is a nutrient that plays an essential role in biological functions. It mediates oxygen transport by hemoglobin and constitutes an essential component of the respiratory chain by conferring redox activity on the cytochromes and other enzymes. However, iron can also damage tissues by catalyzing the conversion of hydrogen peroxide to free-radical ions that attack cellular membranes, proteins and DNA [1, 2]. It is hence not surprising that both iron deficiency and iron overload cause pathologic changes. Disorders of iron homeostasis are among the most common inherited diseases of humans [3, 4]. To tightly control iron homeostasis, a complex network of iron transporters, storage molecules and regulators has evolved. To interface iron metabolism with other metabolic activities of cells, regulators of iron metabolism also respond to non-iron signals such as NO and oxidative stress [5, 6]. Iron homeostasis is regulated at the systemic and at the cellular level. The expression of central proteins involved in iron uptake and transport, iron storage and iron utilization is controlled by the IRE/IRP regulatory system. IREs (iron-responsive elements) are RNA elements that function as binding sites for IRP (iron regulatory protein) -1 and IRP-2. IRP-1 or IRP-2 bound to a single IRE in the 5' untranslated region (UTR) of an mRNA controls the translation of e.g. the iron storage proteins H- and L-ferritin, the erythroid 5aminolevulinate synthase (eALAS) and of mitochondrial aconitase mRNA [7-12]. IRPs bound to multiple IREs in the 3'UTR of the transferrin receptor 1 (TfR1) mRNA stabilize the transcript, which encodes a critical receptor for cellular iron uptake (reviewed in [1315]. The IRE binding activity of IRP-1 and IRP-2 is itself regulated by the experimentally defined “intracellular chelatable iron pool” [16-21]. In addition, H2O2 and nitric oxide 3 From bloodjournal.hematologylibrary.org by guest on June 3, 2013. For personal use only. (NO) affect IRP activity [22-29], linking the regulation of iron metabolism to the oxidative stress and nitric oxide pathways. In addition to IRP-mediated post-transcriptional regulation, transcriptional control mechanisms regulate important aspects of cellular iron homeostasis. For example, the transcription of the transferrin receptor gene is activated by hypoxia inducible factor 1 (HIF-1 ) [30-32] and is downregulated by TNF- and IL-1 in alveolar epithelial cells [33]. L-ferritin mRNA transcription is induced by prostaglandin A1 [34], and H-ferritin transcription is augmented by c-jun in cultured HeLa cells [35]. Both the H-ferritin and IRP-2 genes are targeted by c-myc, and the regulation of these genes is thought to contribute to c-myc-dependent cell proliferation and transformation [36]. The positional cloning of the gene affected in hereditary hemochromatosis (HC) [37] resulted in the identification of a novel protein with a role in iron homeostasis, termed HFE. HC is characterized by systemic iron overload from increased duodenal iron absorption [38]. HFE is an MHC class-1 like protein [37] that forms a heterodimer with 2 microglobulin ( 2M). A missense mutation (C282Y) in the extracellular domain of HFE alters its conformation and abrogates 2M binding, which results in a loss of HFE protein presentation on the cell surface [39, 40]. Other polymorphisms have been found in the HFE gene but their clinical significance is less clear [37, 41, 42]. A biochemical link between HFE and cellular iron metabolism was established with the finding that HFE can engage in high affinity interactions with the transferrin receptor [43, 44]. This interaction interferes with the binding of transferrin to the transferrin receptor, and thus reduces cellular iron uptake [43, 45, 46]. We previously developed a stable HeLa cell line 4 From bloodjournal.hematologylibrary.org by guest on June 3, 2013. For personal use only. in which HFE is expressed under the control of a tetracycline-responsive promoter [47]. We demonstrated that the induction of HFE expression results in decreased iron uptake from diferric transferrin. Moreover, HFE expression activates IRP activity and thus causes reduced synthesis of the iron storage protein ferritin and an increase in transferrin receptor levels [47]. Recently, HFE was overexpressed in Chinese hamster ovary cells [48]. Similar to the observations in HeLa cells [47, 49], HFE overexpression caused a decrease in transferrin-mediated iron uptake. However, the combined expression of both HFE and 2M increased TfR1-dependent iron uptake and cellular iron levels. It was reported that the HFE- 2M complex enhances the rate of TfR1 recycling and results in an increased steady-state level of TfR1 at the plasma membrane of these stably transfected cells [48]. Thus, the availability of 2M may affect HFE function and iron homeostasis. Gene expression profiling using DNA microarrays has allowed to broaden gene expression analyses from studying single genes to investigating complex regulatory networks [50-52]. Here, we report the development of the "IronChip", a cDNA-based microarray that represents human genes directly involved in iron metabolism or in interlinked pathways such as oxidative stress, NO metabolism or copper metabolism. We analyzed the genetic response patterns of HeLa cells to iron perturbation as well as exposure to oxidative stress and the NO+ donor sodiumnitroprusside (SNP). We demonstrate that the resulting regulatory patterns reflect degrees of relationship between the different signals. Remarkably, the gene responses elicited by HFE induction and by pharmacological iron chelation exhibit the highest degree of relatedness, both for IRPand non-IRP target genes. This finding suggests that HFE expression directly targets the regulatory iron pool(s) of the transfected cells. 5 From bloodjournal.hematologylibrary.org by guest on June 3, 2013. For personal use only. Materials and Methods Selection of cDNA clones The genes that are immobilized on the "IronChip" were selected based on (i) literature searches, (ii) microarray experiments performed on filters that contain approximately 20000 human non-redundant ESTs comparing hemin and desferrioxamine treated CaCo2 cells and (iii) gene lists from published microarray studies that address metabolic pathways of interest. 113 human 'expressed sequence tag' (EST) clones, that were sequence verified from both ends were chosen for the "IronChip" (version 2.0) (http://www.embl- heidelberg.de/ExternalInfo/hentze/suppinfo.html.) The ESTs were selected to contain the 3'end of a cDNA (i.e. the polyadenylation signal) and to extend for at least 300bp towards the 5' end. The clone finder software, developed by the HUSAR Biocomputing Service Group at the German Cancer Research Center (http://genome.dkfz-heidelberg.de) facilitated the selection. The clones were purchased from the German Resource Center (RZPD). Preparation of the "IronChip" microarray platform The preparation of the “IronChip” microarray platform, which includes amplification, spotting and attachment of the cDNAs is described elsewhere [53]. The same reference outlines the use of positive and negative hybridization controls integrated into the analysis to determine the cut-off signals for noise as well as the cut-off ratio for differential expression on the "IronChip". 6 From bloodjournal.hematologylibrary.org by guest on June 3, 2013. For personal use only. Synthesis of fluorescent cDNA probes Fluorescent cDNA probes were synthesized from 5 µg total RNA using a linear mRNA amplification protocol, exactly as described in (http://cmgm.stanford.edu/pbrown/protocols/ampprotocol_3.html). 3µg of the T7 RNA polymerase amplified antisense RNA was subsequently subjected to a direct labeling reaction by incorporation of Cy3 and Cy5 fluorescent dyes (Cy3 or Cy5) using random primers (http://cmgm.stanford.edu/pbrown/protocols/4_human_RNA.html; [53]). At least two independent cell culture experiments were performed for each experimental condition tested. Cy3 fluorescent dyes were incorporated into the cDNA synthesized from the control sample and Cy5 fluorescent dyes into cDNA synthesized from the experimental sample and vice versa. This 'dye switch' helps to eliminate technical artifacts that derive from the biophysical properties of the two different dyes. Genes were only scored as differentially expressed if they displayed a consistent regulatory pattern in such dye switch experiments. Microarray analysis The microarrays were immersed at 42°C in 6xSSC/0,5%SDS/1%BSA for 40 min and subsequently washed extensively with ddH2O at room temperature. Prior to hybridization, the spotted PCR products were denatured by immersing the slides at 95°C in ddH2O for 2 min. Excess of liquid was removed from the slides by centrifuging them briefly at 715xg in a microtiter plate centrifuge (Z320, Hermle, Wehingen, Germany). Prior to hybridization, the purified Cy3 and Cy5 labeled cDNAs were mixed, 5 µg poly(dA) and 1 µg human Cot1 DNA (both Gibco Invitrogen Corp., Carlsbad, Ca, USA) were added and subsequently evaporated in a vacuum Concentrator 5301 (Eppendorf, 7 From bloodjournal.hematologylibrary.org by guest on June 3, 2013. For personal use only. Hamburg, Germany) at 60°C. The resulting pellet was dissolved in 12 µl hybridization buffer (50% formamide / 6xSSC / 0,5%SDS / 5x Denhardt) and denatured by incubating at 95°C for 2 min. The probe was then transferred onto the array under a 24x24 mm coverslip and incubated in a humid chamber (GeneMachines, San Carlos, CA, USA) containing 2xSSC drops for providing humidity. Hybridization was performed for 12h to 16h in a 42°C waterbath (GFL, Burgwedel, Germany). After hybridization the microarrays were washed in 0,1xSSC / 0,1%SDS for 10 min and twice with 0,1xSSC for 5 min (on an orbital shaker), followed by a brief immersion of the slides in ddH2O. Finally, the washed slides were dried by centrifuging them briefly at 715xg in a microtiter plate centrifuge (Z320, Hermle, Wehingen, Germany). All washing steps were performed at room temperature. Scanning and data analysis All microarrays were scanned on a GenePix 4000B Microarray Scanner (Axon Instruments, Union City, CA, USA). For each microarray individual laser power and photomultiplier settings were used, allowing all signals to remain in the linear range of the scanner. Separate scan images for Cy3 and Cy5 were produced and analyzed using the ChipSkipper microarray data evaluation software (http://pc-ansorge11.emblheidelberg.de/chipskipper). Intensity values for each spot were calculated by subtraction of the local background surrounding the spot. All spots were used for the calculation of a linear regression line. The regression line’s parameters (offset, slope) were used for normalization. The resulting data were analyzed in Excel (Microsoft Corp., Redmond, WA, USA). At least two independent cell culture experiments were performed for each experimental condition tested and analyzed on the "IronChip" (version 2.0). For the 8 From bloodjournal.hematologylibrary.org by guest on June 3, 2013. For personal use only. bioinformatic analysis of the data, ratios of all the triplicate spots representing one cDNA were averaged. For those genes that are represented by multiple cDNA clones on the "IronChip" the average of ratios of those different clones was calculated. The standard deviation for each resulting ratio was determined. Genes listed in the result tables represent those that have been scored differentially expressed in all the experiments performed for a specific treatment. Genes are scored differentially expressed if the calculated ratios exceed "the ratio cut-off value" defined by the usage of positive spike-in controls [53]. For most experiments this value lies between 1.4- and 1.7- fold. Cell culture, RNA extraction and Northern analysis: The maintenance of cultured HeLa cells and the treatments with 100µM hemin, 100µM ferric ammonium citrate, 100µM desferrioxamine, 100µM H2O2 and 100µM SNP were performed as described previously [27]. All treatments were performed for 8 hours. The establishment and maintenance of the HFE over expressing cell line as well as the experimental conditions of HFE over expression are described in [47]. Total RNA from HeLa cells was extracted using RNAcleanTM (Hybaid-AGS, Heidelberg, Germany) according to manufacturers instructions. For Northern analysis, 10µg of total RNA were separated on a 1% formaldehyde agarose gel and blotted onto a Nylon membrane (Nytran N, Schleicher and Schuell, Dassel, Germany). The membrane was subsequently hybridized to radioactively labeled probes in Church buffer [54]. The signals obtained were quantified on a Fluoroimager (Molecular Dynamics, now Amersham Biosciences, Piscaqtaway, NJ, USA). Sucrose gradient analysis 9 From bloodjournal.hematologylibrary.org by guest on June 3, 2013. For personal use only. The preparation of cytoplasmic extracts from HeLa cells and sucrose gradient centrifugation was described previously [55]. Total RNA was extracted from the sucrose gradient fractions as described in [56]. Results Validation of the human "IronChip" We first established a cDNA microarray platform that represents a selection of human genes which are directly involved in iron metabolism or which play a role in interlinked pathways such as copper metabolism, NO metabolism, the redox pathway, stress responses, selenium metabolism or cell growth (“IronChip”). In addition, we included control genes that are not expected to be affected by the experimental conditions, as well as genes that are not represented in the human genome and hence serve as negative (background) controls and that can be used as so-called spike-in controls for standardization purposes [53]. 113 different human genes represented by up to three independent cDNA clones were selected for the version 2.0 of the "IronChip". The names of these genes and their corresponding Genbank accession numbers are shown at http://www.embl-heidelberg.de/ExternalInfo/hentze/suppinfo.html. In addition, Genebank accession numbers are included in the text for all mentioned “IronChip” genes. To assess whether the "IronChip" reflects changes in mRNA levels in response to iron perturbations, HeLa cells were either iron loaded by treatment with 100µM hemin (H) for 8 hours, or made iron-deficient by incubation with 100µM desferrioxamine (D) for 8 hours. Total RNA was purified from the treated cells, labeled with Cy3 (D) and Cy5 (H), or vice versa (see Materials and Methods), and analyzed on the "IronChip". The results of 10 From bloodjournal.hematologylibrary.org by guest on June 3, 2013. For personal use only. these experiments are shown in figure 1a-d. As expected, TfR1 (NM_003234) mRNA levels are increased in iron deficient cells, consistent with a stabilization of the TfR1 mRNA by IRP binding to its 3'UTR [57, 58]. We further observe an approximately twofold increase in the IRE-containing splice variant of DMT1/ DCT1/Nramp2 (AB004857) mRNA, consistent with the notion that IRP binding to the IRE in the 3'UTR stabilizes this mRNA [59]. In hemin-treated cells, we observe a strong increase of heme oxygenase-1 (HO-1) (X06985) mRNA, that encodes a critical enzyme in heme breakdown [60]. This result confirms earlier findings in cultured pig alveolar macrophages and in a human leukemia cell line [61, 62]. L-ferritin (M11147) mRNA expression is also increased in hemin-treated cells, while H-ferritin (M11146) mRNA levels remain unchanged. A comparable result was obtained in rat liver after iron administration [56]. House keeping genes, like glyceraldehyde phosphate dehydrogenase (GAPDH) (M33197) or -actin (X00351) are not affected (fig.1a, b and 1d). These results show that the microarray analysis on the “IronChip” accurately reflects the cellular responses to iron perturbations that have been observed earlier. To further validate this approach, Northern blots were performed for eight selected genes and quantitated by phosphoimaging (fig. 1d). The close correlation between the results obtained by microarray analysis and Northern blotting confirms that the “IronChip” provides a reliable tool for the qualitative and quantitative analysis of gene expression in human iron metabolism. In addition to those genes that are directly involved in iron metabolism, we found some additional genes to be regulated. In iron replete cells, three members of the heat shock protein (hsp) family [hsp70D (M11717), hsp105 ( 11 003334) and the mitochondrial From bloodjournal.hematologylibrary.org by guest on June 3, 2013. For personal use only. (m) hsp70 (L11066)] and three genes mediating growth effects [c-myc (V00568) and growth arrest specific (gas)- 1 (L13698) and 3 (L03203)] show increased mRNA levels. In iron deficient cells, we observe a robust increase in the amount of mRNAs encoding metallothionine (mt)-2 (X97260), lysyl oxidase (lox; M94054), and a small but consistent increase in the mRNA level of hypoxia inducible factor HIF-1 (NM_001530). Furthermore, the expression of c-jun (J04111), a gene involved in cell proliferation is increased in iron deficient cells (fig. 1c). Note, that the quantitative changes in gene expression in this experiment are monitored as the sum of increased and decreased expression for a given gene in both conditions tested. Gene expression profiles derived from hemin and ferric ammonium citrate (FAC) treated HeLa cells. We next assessed HeLa cells that were treated with two different sources of iron, hemin (ferric protoporphyrin IX) or ferric ammonium citrate (FAC), to address two questions: first, whether the iron loaded state resulting from both treatments elicited a common pattern in the respective expression profiles; second, whether these two similar treatments could be discriminated by diagnostic features of the respective gene response patterns. Subconfluent HeLa cells were treated either with 100µM hemin or 100µM FAC for 8 hours. Untreated HeLa cells were used as a control for both. As can be seen in figure 2, the expression profiles derived from hemin and FAC-treated cells closely resemble each other. Genes that are differentially expressed after the treatment with both iron sources include HO-1, Hsp70D, mhsp70, L-ferritin, gas3, TfR-1 and mt-2. The increased expression of the first five and the decreased expression of the latter two genes appears to 12 From bloodjournal.hematologylibrary.org by guest on June 3, 2013. For personal use only. define a common denominator that hallmarks the iron loaded state. In general, the magnitude of the expression change is lower in the FAC-treated cells. Note that the induction of HO-1 mRNA is highly pronounced in hemin-treated cells, consistent with the function of HO-1 in heme breakdown [63]. Furthermore, a more than 1.4-fold change in mRNA levels (which we utilized as the minimum defining cut-off between ‘regulated’ and ‘unregulated’ genes; see Materials and Methods) of hsp105 , c-jun, lysyl oxidase, gas-1 and Hif-1 mRNAs was unique to the hemin-treated cells and not observed following FAC administration. These data show that microarray analysis with the "IronChip" can identify both the common features that identify cellular iron load as well as the distinct features that allow the discrimination between the sources of iron. Gene expression profiles of H2O2- and sodium nitroprusside (SNP)- treated HeLa cells H2O2 treatment and iron deficiency both activate IRP-1 [22-28] and trigger posttranscriptional changes in the expression of IRE-regulated mRNAs. As a consequence, H-and L-ferritin mRNA translation is repressed, and transferrin receptor mRNA levels increase in both conditions [64]. We next recorded the broader gene expression profile from H2O2-treated HeLa cells and assessed whether it can be distinguished from the gene expression profile derived from iron-deficient cells. HeLa cells were exposed to 100µM H2O2 for 8 hours and total RNA was subsequently analyzed on the "IronChip" in comparison to total RNA from untreated control cells. H2O2 treatment induced increased HO-1 and TfR-1 mRNA levels (fig.3a). The induction of HO-1 by H2O2 has been reported previously [65]. By contrast, we neither observe the regulation of the IRE-containing DMT1/ DCT1/Nramp2 mRNA, nor any regulation of 13 From bloodjournal.hematologylibrary.org by guest on June 3, 2013. For personal use only. those genes that are seen regulated in iron-deficient HeLa cells (fig.4a). Thus the expression profile derived from H2O2 treated HeLa cells is clearly distinct from the gene expression profiles obtained from iron-deficient HeLa cells. In addition to iron perturbation and oxidative stress, also nitric oxide affects IRP activity and the regulation of IRE-containing mRNAs [22, 24, 25, 27, 28]. Thus, we also tested the effect of sodium nitroprusside (SNP), which releases nitrosonium ions (NO+), on the regulation of the genes immobilized on the "IronChip". NO+ has been suggested to cause the S-nitrosylation of critical thiol groups, to prevent the binding of IRP-2 to IREs, and to result in TfR-1 mRNA degradation [66]. HeLa cells were treated with 100µM SNP for 8 hours; total RNA was extracted and analyzed on the "IronChip" in comparison to an untreated control sample. As expected, SNP treatment reduces TfR mRNA levels (fig 3b). In addition, the mRNA levels of the IRE-containing splice variant DMT1/DCT1/Nramp2 are also reduced. The approximately two-fold increase of DMT1/DCT1/Nramp2 mRNA in iron deficiency (when compared to hemin- treated cells; fig. 1) and its reduced expression in response to SNP is consistent with an IRP mediated regulatory mechanism of DCT1/DMT1/Nramp2 mRNA stability. In contrast to iron manipulated HeLa cells, hsp70D is co regulated with TfR-1 and DCT1 in SNP-treated cells. SNP treatment strongly induces HO-1 and affects the expression of the metallothionines 1 and 2 (fig.3b). The regulation of heterogeneous nuclear ribonucleoprotein D-like protein JKTBP (D89092) and of the prion protein (M13899) detected after SNP treatment of HeLa cells was not observed in iron perturbed or H2O2 treated HeLa cells. With the exception of IRP target genes, the expression profile 14 From bloodjournal.hematologylibrary.org by guest on June 3, 2013. For personal use only. obtained in SNP-treated HeLa cells is clearly distinct from those of iron manipulated or H2O2-treated HeLa cells. HFE expression and iron deficiency yield highly similar gene expression profiles Previous work showed that induced HFE expression in mammalian cells resulted in decreased iron uptake from diferric transferrin, IRP activation, and the regulation of IRP target mRNAs [43, 46, 47, 67-70]. These findings suggested that HFE expression in transfected cells affects the regulatory "labile iron pool" in a way that is similar to desferrioxamine-induced iron starvation. To record the responses of transfected HeLa cells to induced HFE expression more globally and to compare these with the response of desferrioxamine-treated cells, additional microarray analyses were performed. HeLa cells were stably transfected with the human HFE cDNA under the control of a doxycyclin-responsive promoter [47]. The absence of doxycyclin induces HFE expression [47], whereas the transgene is not transcribed following the addition of doxycyclin to the culture medium. Total RNA extracted from doxycyclin-treated and untreated HeLa cells bearing the HFE transgene was used for fluorescent cDNA synthesis and subsequent analysis on the "IronChip". As expected, the HFE mRNA is strongly induced in the absence of doxycyclin (fig.4a). When HFE expression is induced, TfR-1, c-jun, lysyl oxidase and Mt-2 mRNAs are increased, whereas the mRNA levels of HO-1, Hsp70D, Hsp105a, mHsp70, L-Fer and Gas-3 decrease. These data were confirmed by northern analysis (fig.4b). Doxycyclin treatment of non-transfected HeLa cells did not affect the regulation of "IronChip" genes (data not shown). When HeLa cells were treated with 100µM desferrioxamine, TfR-1, c-jun and lysyl oxidase mRNA levels increased. 15 From bloodjournal.hematologylibrary.org by guest on June 3, 2013. For personal use only. Decreased mRNA levels were found for HO-1, Hsp70D, Hsp105a, mHsp70, L-Fer, Gas 1 and Gas 3 and c-myc (Fig.4a). These data sets reveal striking similarities between the gene responses elicited by HFE expression and desferrioxamine treatment, respectively. This conclusion applies both to IRP-target genes and non-IRP target genes (fig.4c). Both gene response patterns significantly differ from those elicited by e.g. SNP and H2O2 treatment (fig 3). Thus, we conclude that HFE expression in transfected HeLa cells triggers cellular iron deficiency. Monitoring translational responses to iron perturbations by microarray analyses Traditionally, microarray analyses are employed to monitor changes in steady state mRNA levels. Because gene regulation in response to iron perturbations also prominently involves translational control in the absence of concomitant changes in mRNA levels, we wanted to adapt our experimental approach to reveal regulation at the translational level. Cytoplasmic extracts were prepared from HeLa cells that were treated with either hemin or desferrioxamine, and subjected to linear sucrose gradient centrifugation (see Materials and Methods). Six fractions were prepared from each sample, total RNA was extracted and initially analyzed by Northern blotting. As shown in fig.5a, L-ferritin mRNA is enriched in the polysomal fractions at the bottom of the sucrose gradient in hemin-treated cells, as previously observed [56]. In iron deficient cells, L-ferritin mRNA is enriched in the fractions that contain monosomes (80S) and mRNPs, consistent with previous findings that IRP inhibits the translation of ferritin mRNAs by interfering with the first steps of the translation initiation process [71]. As a control, actin mRNA remains localized in the polysomal fractions in both hemin-and desferrioxamine-treated cells.. 16 From bloodjournal.hematologylibrary.org by guest on June 3, 2013. For personal use only. For microarray analyses, we pooled the total RNA derived from the polysomal fractions 1-3 and the total RNA derived from the monosomal/mRNP fractions 4-5, respectively. For each condition (hemin and desferrioxamine treatment), the polysomal and monosomal/mRNP fractions were labeled with different fluorescent dyes and hybridized to the "IronChip". Similar to the Northern blots, the data (fig. 5b) clearly reveal the translational regulation of L-ferritin mRNA and the lack of actin mRNA regulation. Similar to L-ferritin, the “IronChip” reflects also the translational control of the H-ferritin mRNA in response to iron perturbation (fig.5b). Mitochondrial aconitase mRNA and eALAS mRNA which have also been shown to be translationally regulated by an IRE in their mRNAs [9, 11] are not expressed at sufficiently high levels in HeLa cells to allow a reliable assessment of their ribosome association. Likewise, the IRE-containing iron transporter IREG-1/ferroportin/MTP-1 is mainly expressed in duodenal enterocytes, macrophages and the placenta [72-74], and its mRNA is undetectable in HeLa cells. No additional genes represented on the "IronChip" (version 2.0) show an altered translation of their mRNAs. We conclude that microarray analyses with the “IronChip” can also be used to monitor iron-induced changes in mRNA translation. Discussion We have studied the genetic responses of a human cell line to changes in iron metabolism employing a newly developed cDNA-based microarray platform (“IronChip”). Using this approach, novel insights into human iron metabolism were obtained (see below). In addition, the results show the utility of the “IronChip” as a versatile tool to investigate a broad range of questions regarding the physiology of human iron metabolism and diseases that result from its aberrations. 17 From bloodjournal.hematologylibrary.org by guest on June 3, 2013. For personal use only. New insights into human iron metabolism Human HeLa cells have served as an intensively characterized model system for the investigation of iron metabolism. We therefore chose HeLa cells to explore the utility of a specialized DNA microarray that represents 113 different human genes and that was expected to reveal insights into regulatory responses of human cells to iron deficiency, iron overload, HFE expression and small signaling molecules. Table 1 provides a synopsis over these responses. Importantly, for the genes known to be regulated by iron, the microarray data are consistent with the existing literature. Moreover, many of the emerging results were confirmed by Northern blotting (figs 1d and 4b), which in addition ascertained a surprisingly good performance of the microarray platform in yielding quantitatively accurate data. Heme oxygenase (HO)-1 emerges as the most strongly responsive gene in our dataset. HO-1 mRNA levels decrease in iron-deficient and in HFE-expressing cells, and increase in response to iron loading as well as SNP and H2O2 exposure (table 1). HO-1 may thus represent a central ‘stress response gene’ in the iron regulatory network. Hsp70D appeared as another strongly responsive gene. Although it did not show regulation in H2O2-treated cells, it responded to all other experimental perturbations. It is conceivable that additional genes (perhaps including Hsp70D) might have responded to higher concentrations of H2O2, or if a different cell line had been tested. Only two genes displayed H2O2 regulation under our experimental conditions (table 1). However, Hsp70D mRNA levels responded to iron deficiency (and to HFE expression) and iron overload more strongly than TfR1 mRNA levels, which are often considered to be the 18 From bloodjournal.hematologylibrary.org by guest on June 3, 2013. For personal use only. classic regulatory response to these challenges. It is also notable that Gas 3 and mHsp70 are consistently regulated by altered cellular iron supply. The latter result establishes mitochondrial Hsp70 (mHsp70) as a human iron-responsive gene and reveals that this regulation appears to be conserved between man and yeast: yeast mutants (ssc2-1) of mHsp70 show increased cellular iron uptake and the excess iron accumulates in the mitochondria [75]. It will be important to explore whether the induction of mHsp70 in iron-loaded cells fulfills a protective or a regulatory function in human cells. Previous studies indicated that the heterologous or induced expression of the HFE protein negatively affects cellular iron uptake via the transferrin receptor [43, 46, 47, 67-70] and hence triggers an iron deficiency response by the IRE/IRP regulatory network [47, 69]. We find that desferrioxamine treatment and the induction of HFE expression, respectively, yield nearly identical responses of the 113 genes represented on the "IronChip". This allows the conclusion that HFE expression in this experimental system not only triggers an iron deficiency response by the IRE/IRP network. Rather, the iron deficiency state induced by HFE affects every regulatory system that is also reached by desferrioxamine [76-78]. Since the list of regulated genes includes non-IRP target genes such as the growth effect genes (e.g. c-jun, Gas3) and stress response genes (e.g. HO-1, Hsp70D,Hsp105 , mHSP70), we suspect that at least a part of these responses may be mediated transcriptionally. Regarding the putative function of HFE as an inducer of cellular iron deficiency, one needs to consider that we expressed HFE without induced co-expression of its heterodimerization partner 2 microglobulin. It has been reported that the co-expression of both yields opposite effects to the induction of HFE alone [48]. From a more methodological perspective, the close resemblance of the desferrioxamine19 From bloodjournal.hematologylibrary.org by guest on June 3, 2013. For personal use only. and HFE-induced gene regulatory responses highlights an important application of microarray analyses in studying iron metabolism: the identification of similar profiles elicited by two different stimuli allows to reveal common effector functions. The similarity between the genetic responses to iron overload by hemin and ferric ammonium citrate (FAC), respectively, was predicted and has been confirmed with the “IronChip” (fig. 2). Most genes that respond to FAC also respond to hemin, and the latter response is usually slightly stronger. As an exception to this, the HO-1 mRNA response to hemin is far stronger than to FAC. Considering the biological role of HO-1 in heme breakdown, a more profound induction of HO-1 mRNA by hemin is not surprising. We suggest that the magnitude of the HO-1 response in relation to the responses by mHsp70, L-fer, Gas3, TfR1 and Mt-2 is “diagnostic” for hemin-induced versus FAC-induced iron overload. More generally, this analysis provides an example for the possibility to discriminate between two related stimuli by “IronChip analysis”. The “IronChip” provides a versatile tool for the analysis of iron metabolism As illustrated above, the human “IronChip” was validated as a reliable assay system to identify the responses of more than a hundred genes involved in iron metabolism and interlinked biological pathways. We also show that in combination with sucrose gradient analysis, the “IronChip” successfully identifies genetic responses at the translational level (fig. 5). This is particularly pertinent for the study of mammalian iron metabolism [13]. Compared to far more comprehensive cDNA or oligonucleotide-based microarrays, the “IronChip” offers only limited chances to identify “new genes” that are regulated by 20 From bloodjournal.hematologylibrary.org by guest on June 3, 2013. For personal use only. particular stimuli. For this reason, we believe that more comprehensive microarrays can offer helpful entry points for microarray studies and help to identify genes to be included on the “IronChip”, as was done during the original design and is being used for updated versions (see below). On the other hand, we believe that both gene verification and technical performance parameters of the "IronChip" compare favorably with those of larger arrays. All the genes represented on our arrays have been sequence verified from both ends. Due to the limited number of genes, each gene can be spotted multiple times and in different locations of the chip, and many genes are represented by up to three independent cDNA clones. This redundancy offers additional controls for gene specificity. A major application of the “IronChip” lies in the identification of gene regulatory patterns that provide a characteristic “fingerprint” of a particular treatment or genetic alteration. For this application, the technical quality of the data is critical, particularly the ability to score limited quantitative differences reliably and reproducibly. The recognition of similarities or differences in the genetic responses to different stimuli can be highly informative, and we suggest that the “IronChip” could also prove useful in the analysis of human patient samples. Recently, we increased the number of different relevant genes that are represented on the “IronChip” to nearly 300 (version 3.0) (data not shown). This should further enhance its utility in defining precise gene response patterns and hence to ultimately understand the networks that operate within human iron metabolism. Moreover, we also established an analogous microarray platform with murine cDNAs (data not shown). The murine “IronChip” will not only facilitate cross-species comparisons, but in particular facilitate access to the growing pool of genetic murine 21 From bloodjournal.hematologylibrary.org by guest on June 3, 2013. For personal use only. model systems for human diseases of iron metabolism and to integrate findings from animal models into our understanding of human iron physiology and pathophysiology. Figure legends: Figure 1: Gene expression profiles from iron manipulated HeLa cells. HeLa cells were treated with 100µM hemin (H) or with 100µM desferrioxamine (D) for 8 hours and total RNA was purified from the cells. Fluorescent probes synthesized from total RNA derived from hemin treated cells were labeled with Cy5-modified dUTPs and those synthesized from total RNA derived from desferrioxamine treated cells were labeled with Cy3-modified dUTPs and analyzed on the "IronChip". A) Virtual "IronChip". Colors correspond to the calculated compensated ratios. Red spots represent genes with increased mRNA levels in hemin-treated cells. Green spots represent genes with increased mRNA levels in desferrioxamine-treated cells. Yellow spots represent genes that are equally expressed in both conditions tested. Selected genes are annotated. (B) Scatter Plot analysis. Signals corresponding to the desferrioxamine-treated sample are represented on the y-axis. Signals corresponding to the hemin-treated sample are represented on the x-axis. In the experiment shown here a gene is considered to be differentially expressed, if the H/D ratio is calculated above 1.4 or below 0.7. Genes with a calculated H/D ratio >1.4 (1.4 fold) are shown in red. Genes with a calculated H/D ratio <0.7 (-1.4 fold) are represented in green. House keeping genes, like GAPDH and actin are represented in yellow and negative controls are shown in blue. Positive spike-in controls [53], that have been added in equal amounts to the total RNA of hemin- and desferrioxamine-treated cells and thus by definition should not appear regulated are 22 From bloodjournal.hematologylibrary.org by guest on June 3, 2013. For personal use only. shown in pink. (C) Data table: The average ratios of differentially expressed genes in hemin- (H) and desferrioxamine- (D) treated cells (H/D) are indicated. The standard deviations are shown. (D) Northern blot analysis of selected mRNAs. The ratios of signals obtained in H and D treated cells (as quantified on a Fluoroimager) are indicated. Figure 2: Gene expression profiles derived from hemin and ferric ammonium citrate (FAC) treated HeLa cells. (A) HeLa cells were treated with 100µM hemin (H) or 100µM ferric ammonium citrate (FAC) for 8h. Total RNA was extracted and analyzed on the "IronChip" in comparison to an untreated control sample. The average ratios of differentially expressed genes are indicated with their respective standard deviations. (B) Comparison of the gene expression profiles of hemin and FAC treated HeLa cells. Genes, which show increased expression in hemin and/or FAC treated cells are shown in positive numbers and those with decreased expression in negative numbers. Figure 3: Gene expression profiles of H2O2- and sodium nitroprusside (SNP)treated HeLa cells (A) HeLa cells were treated with 100µM H2O2 for 8h, total RNA was extracted and analyzed on the "IronChip" in comparison to an untreated control sample. Average ratios of differentially expressed genes and their standard deviations are shown. (B) HeLa cells were treated with 100µM SNP for 8h, total RNA was extracted and analyzed on the "IronChip". The average ratios of genes that differ in their expression levels in comparison to untreated control cells are shown. The standard deviations are indicated. 23 From bloodjournal.hematologylibrary.org by guest on June 3, 2013. For personal use only. Figure 4: Gene expression profiles obtained from desferrioxamine-treated HeLa cells and HFE expressing HeLa cells. (A) Total RNA derived from HeLa cells treated with 100µM desferrioxamine (D) for 8h or total RNA derived from HeLa cells that express HFE from an inducible transgene was analyzed on the "IronChip" in comparison to respective control samples. The average ratios of differentially expressed genes and their respective standard deviations are shown. (B) Comparison of the gene expression profiles from desferrioxamine-treated and HFE expressing HeLa cells. Genes with increased expression in desferrioxamine treated and/or HFE expressing cells are shown in positive numbers and those with decreased expression in negative numbers. Figure 5: Microarray assessment of iron-mediated translational control (A) Cytoplasmic extracts from hemin and desferrioxamine treated HeLa cells were sedimented through a 10-40% sucrose gradient (see Material and Methods). The profile on the top denotes the A254 absorption profile. The positions of polysomes, monosomes (80S) and mRNPs are indicated. Northern blot analysis was performed with total RNA extracted from the six individual fractions obtained from the sucrose gradient. The Northern blot was sequentially probed with radiolabeled probes for actin and L-ferritin. The signals obtained from the individual fractions were quantified on Fluoroimager (Molecular Dynamics) and the signals in the polysomes and the 80S and mRNP fractions were calculated as a percentage of the sum of signals in all lanes. The ratio between the 80S and mRNP (mRNP) fractions and the polysomal fractions (PS) is indicated. (B) The three fractions containing polysomal (PS) and the three fractions containing the monosomal and mRNP-derived RNA, respectively, were pooled for each condition and 24 From bloodjournal.hematologylibrary.org by guest on June 3, 2013. For personal use only. analyzed on the "IronChip". The regulatory ratio between mRNP and polysomal fractions is indicated for the L-ferritin (L-fer), H-ferritin (H-fer), actin and gapdh. Table 1: Summary of regulatory responses The regulatory profiles of desferrioxamine- (D) treated, Hfe expressing (Hfe), ferric ammonium citrate- (FAC), hemin-, sodium nitroprusside (SNP)- and H202-treated HeLa cells are summarized. All genes that were scored differentially expressed in the different treatments are listed. (red) indicates a more than 3 fold decrease in mRNA levels. (red) indicates a decrease in mRNA levels between at least 1.4 fold and 2.9 fold. (green) indicates a more than 3 fold increase in mRNA levels. (green) indicates an increase in mRNA levels between at least 1.4 fold and 2.9 fold. (grey) indicates no significant change in mRNA levels. Literatur 1.Aisen P, Enns C, Wessling-Resnick M. Chemistry and biology of eukaryotic iron metabolism. Int J Biochem Cell Biol. 2001; 33: 940-959. 2.Andrews NC. Metal transporters and disease. Curr Opin Chem Biol. 2002; 6: 181-186. 3.Anderson GJ. Ironing out disease: inherited disorders of iron homeostasis. IUBMB Life. 2001; 51: 11-17. 4.Andrews NC. Disorders of iron metabolism. N Engl J Med. 2000; 341: 1986-95 25 From bloodjournal.hematologylibrary.org by guest on June 3, 2013. For personal use only. 5.Cairo G, Recalcati S, Pietrangelo A, Minotti G. The iron regulatory proteins: targets and modulators of free radical reactions and oxidative damage. Free Radic Biol Med. 2002; 32: 1237-1243. 6.Hentze MW, Kühn LC. Molecular control of vertebrate iron metabolism: mRNA-based regulatory circuits operated by iron, nitric oxide, and oxidative stress. Proc Natl Acad Sci U S A. 1996; 93: 8175-8182. 7.Cox TC, Bawden MJ, Martin A, May BK. Human erythroid 5-aminolevulinate synthase: promoter analysis and identification of an iron-responsive element in the mRNA. Embo J. 1991; 10: 1891-1902. 8.Dandekar T, Stripecke R, Gray NK, et al. Identification of a novel iron-responsive element in murine and human erythroid delta-aminolevulinic acid synthase mRNA. Embo J. 1991; 10: 1903-1909. 9.Gray NK, Pantopoulos K, Dandekar T, Ackrell BA, Hentze MW. Translational regulation of mammalian and Drosophila citric acid cycle enzymes via iron-responsive elements. Proc Natl Acad Sci U S A. 1996; 93: 4925-4930. 10.Hentze MW, Caughman SW, Rouault TA, et al. Identification of the iron-responsive element for the translational regulation of human ferritin mRNA. Science. 1987; 238: 1570-1573. 26 From bloodjournal.hematologylibrary.org by guest on June 3, 2013. For personal use only. 11.Kim HY, LaVaute T, Iwai K, Klausner RD, Rouault TA. Identification of a conserved and functional iron-responsive element in the 5'-untranslated region of mammalian mitochondrial aconitase.J Biol Chem. 1996; 271: 24226 -24230. 12.Kohler SA, Henderson BR, Kuhn LC. Succinate dehydrogenase b mRNA of Drosophila melanogaster has a functional iron-responsive element in its 5'-untranslated region. J Biol Chem. 1995; 270: 30781-30786. 13.Muckenthaler M, Hentze MW. Mechanisms for posttranscriptional regulation by iron-responsive elements and iron regulatory proteins. Prog Mol Subcell Biol. 1997; 18: 93-115. 14.Cairo G, Pietrangelo A. Iron regulatory proteins in pathobiology. Biochem J. 2000; 352: 241-250. 15.Eisenstein RS. Iron regulatory proteins and the molecular control of mammalian iron metabolism. Annu Rev Nutr. 2000; 20: 627-662. 16.Guo B, Yu Y, Leibold EA. Iron regulates cytoplasmic levels of a novel ironresponsive element- binding protein without aconitase activity. J Biol Chem. 1994; 269: 24252-24260. 27 From bloodjournal.hematologylibrary.org by guest on June 3, 2013. For personal use only. 17.Haile DJ, Hentze MW, Rouault TA, Harford JB, Klausner RD. Regulation of interaction of the iron-responsive element binding protein with iron-responsive RNA elements. Mol Cell Biol. 1989; 9: 5055-5061. 18.Henderson BR, Seiser C, Kühn LC. Characterization of a second RNA-binding protein in rodents with specificity for iron-responsive elements. J Biol Chem. 1993; 268: 27327-27334. 19.Pantopoulos K, Gray NK, Hentze MW. Differential regulation of two related RNAbinding proteins, iron regulatory protein (IRP) and IRPB. Rna. 1995; 1: 155-163. 20.Rouault TA, Hentze MW, Caughman SW, Harford JB, Klausner RD. Binding of a cytosolic protein to the iron-responsive element of human ferritin messenger RNA. Science. 1988; 241: 1207-1210. 21.Samaniego F, Chin J, Iwai K, Rouault TA, Klausner RD. Molecular characterization of a second iron-responsive element binding protein, iron regulatory protein 2. Structure, function, and post- translational regulation. J Biol Chem. 1994; 269: 30904-30910. 22.Drapier JC, Hirling H, Wietzerbin J, Kaldy P, Kühn LC. Biosynthesis of nitric oxide activates iron regulatory factor in macrophages. Embo J. 1993; 12: 3643-3649. 28 From bloodjournal.hematologylibrary.org by guest on June 3, 2013. For personal use only. 23.Martins EA, Robalinho RL, Meneghini R. Oxidative stress induces activation of a cytosolic protein responsible for control of iron uptake. Arch Biochem Biophys. 1995; 316: 128-134. 24.Oria R, Sanchez L, Houston T, Hentze MW, Liew FY, Brock JH. Effect of nitric oxide on expression of transferrin receptor and ferritin and on cellular iron metabolism in K562 human erythroleukemia cells. Blood. 1995; 85: 2962-2966. 25.Pantopoulos K, Hentze MW. Nitric oxide signaling to iron-regulatory protein: direct control of ferritin mRNA translation and transferrin receptor mRNA stability in transfected fibroblasts. Proc Natl Acad Sci U S A. 1995; 92: 1267-1271. 26.Pantopoulos K, Hentze MW. Activation of iron regulatory protein-1 by oxidative stress in vitro. Proc Natl Acad Sci U S A. 1998; 95: 10559-10563. 27.Pantopoulos K, Weiss G, Hentze MW. Nitric oxide and oxidative stress (H2O2) control mammalian iron metabolism by different pathways. Mol Cell Biol. 1996; 16: 3781-3788. 28.Richardson DR, Neumannova V, Nagy E, Ponka P. The effect of redox-related species of nitrogen monoxide on transferrin and iron uptake and cellular proliferation of erythroleukemia (K562) cells. Blood. 1995; 86: 3211-3219. 29 From bloodjournal.hematologylibrary.org by guest on June 3, 2013. For personal use only. 29.Weiss G, Goossen B, Doppler W, et al. Translational regulation via iron-responsive elements by the nitric oxide/NO-synthase pathway. Embo J. 1993; 12: 3651-3657. 30.Bianchi L, Tacchini L, Cairo G. HIF-1-mediated activation of transferrin receptor gene transcription by iron chelation. Nucleic Acids Res. 1999; 27: 4223-4227. 31.Lok CN, Ponka P. Identification of a hypoxia response element in the transferrin receptor gene. J Biol Chem. 1999; 274: 24147-24152. 32.Tacchini L, Bianchi L, Bernelli-Zazzera A, Cairo G. Transferrin receptor induction by hypoxia. HIF-1-mediated transcriptional activation and cell-specific post-transcriptional regulation. J Biol Chem. 1999; 274: 24142-24146. 33.Smirnov IM, Bailey K, Flowers CH, Garrigues NW, Wesselius LJ. Effects of TNFalpha and IL-1beta on iron metabolism by A549 cells and influence on cytotoxicity. Am J Physiol. 1999; 277: L257-263. 34.Elia G, Polla B, Rossi A, Santoro MG. Induction of ferritin and heat shock proteins by prostaglandin A1 in human monocytes. Evidence for transcriptional and posttranscriptional regulation. Eur J Biochem. 1999; 264: 736-745. 35.Faniello MC, Chirico G, Quaresima B, et al. An alternative model of H ferritin promoter transactivation by c-Jun. Biochem J. 2002; 363: 53-58. 30 From bloodjournal.hematologylibrary.org by guest on June 3, 2013. For personal use only. 36.Wu KJ, Polack A, Dalla-Favera R. Coordinated regulation of iron-controlling genes, H-ferritin and IRP2, by c-MYC. Science. 1999; 283: 676-679. 37.Feder JN, Gnirke A, Thomas W, et al. A novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis. Nat Genet. 1996; 13: 399-408. 38.Sheth S, Brittenham GM. Genetic disorders affecting proteins of iron metabolism: clinical implications. Annu Rev Med. 2000; 51: 443-464. 39.Feder JN, Tsuchihashi Z, Irrinki A, et al. The hemochromatosis founder mutation in HLA-H disrupts beta2- microglobulin interaction and cell surface expression. J Biol Chem. 1997; 272: 14025-14028. 40.Waheed A, Parkkila S, Zhou XY, et al. Hereditary hemochromatosis: effects of C282Y and H63D mutations on association with beta2-microglobulin, intracellular processing, and cell surface expression of the HFE protein in COS-7 cells. Proc Natl Acad Sci U S A. 1997; 94: 12384-12389. 41.Bradbury R, Fagan E, Payne SJ. Two novel polymorphisms (E277K and V212V) in the haemochromatosis gene HFE. Hum Mutat. 2000; 15: 120. 42.Douabin V, Moirand R, Jouanolle A, et al. Polymorphisms in the HFE gene. Hum Hered. 1999; 49: 21-26. 31 From bloodjournal.hematologylibrary.org by guest on June 3, 2013. For personal use only. 43.Feder JN, Penny DM, Irrinki A, et al. The hemochromatosis gene product complexes with the transferrin receptor and lowers its affinity for ligand binding. Proc Natl Acad Sci U S A. 1998; 95: 1472-1477. 44.Parkkila S, Waheed A, Britton RS, et al. Association of the transferrin receptor in human placenta with HFE, the protein defective in hereditary hemochromatosis. Proc Natl Acad Sci U S A. 1997; 94: 13198-13202. 45.Lebron JA, Bjorkman PJ. The transferrin receptor binding site on HFE, the class I MHC-related protein mutated in hereditary hemochromatosis. J Mol Biol. 1999; 289: 1109-1118. 46.Roy CN, Penny DM, Feder JN, Enns CA. The hereditary hemochromatosis protein, HFE, specifically regulates transferrin-mediated iron uptake in HeLa cells. J Biol Chem. 1999; 274: 9022-9028. 47.Riedel HD, Muckenthaler MU, Gehrke SG, et al. HFE downregulates iron uptake from transferrin and induces iron- regulatory protein activity in stably transfected cells. Blood. 1999; 94: 3915-3921. 48.Waheed A, Grubb JH, Zhou XY, et al. Regulation of transferrin-mediated iron uptake by HFE, the protein defective in hereditary hemochromatosis. Proc Natl Acad Sci U S A. 2002; 99: 3117-3122. 32 From bloodjournal.hematologylibrary.org by guest on June 3, 2013. For personal use only. 49.Gross CN, Irrinki A, Feder JN, Enns CA. Co-trafficking of HFE, a nonclassical major histocompatibility complex class I protein, with the transferrin receptor implies a role in intracellular iron regulation. J Biol Chem. 1998; 273: 22068-22074. 50.Kallioniemi OP. Biochip technologies in cancer research. Ann Med. 2001; 33: 142147. 51.Schulze A, Downward J. Navigating gene expression using microarrays--a technology review. Nat Cell Biol. 2001; 3: 190-195. 52.Eide DJ. Functional genomics and metal metabolism. Genome Biol. 2001; 2: 1-3. 53.Richter A, Schwager C, Hentze S, Ansorge W, Hentze M, Muckenthaler M. Comparison of fluorescent tag DNA labeling methods used for expression analysis by DNA microarrays. Biotechniques. 2002; (in press). 54.Sambrook J, Fritsch EF, Maniatis T, Molecular Cloning: A laboratory manual. 1989, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory. 55.Körner CG, Wormington M, Muckenthaler M, Schneider S, Dehlin E, Wahle E. The deadenylating nuclease (DAN) is involved in poly(A) tail removal during the meiotic maturation of Xenopus oocytes. Embo J. 1998; 17: 5427-5437. 33 From bloodjournal.hematologylibrary.org by guest on June 3, 2013. For personal use only. 56.White K, Munro HN. Induction of ferritin subunit synthesis by iron is regulated at both the transcriptional and translational levels. J Biol Chem. 1988; 263: 8938-8942. 57.Casey JL, Koeller DM, Ramin VC, Klausner RD, Harford JB. Iron regulation of transferrin receptor mRNA levels requires iron- responsive elements and a rapid turnover determinant in the 3' untranslated region of the mRNA. Embo J. 1989; 8: 36933699. 58.Müllner EW, Neupert B, Kühn LC. A specific mRNA binding factor regulates the iron-dependent stability of cytoplasmic transferrin receptor mRNA. Cell. 1989; 58: 373382. 59.Gunshin H, Allerson CR, Polycarpou-Schwarz M, et al. Iron-dependent regulation of the divalent metal ion transporter. FEBS Lett. 2001; 509: 309-316. 60.Tenhunen R, Marver HS, Schmid R. Microsomal heme oxygenase. Characterization of the enzyme. J Biol Chem. 1969; 244: 6388-6394. 61.Hoffman R, Ibrahim N, Murnane MJ, Diamond A, Forget BG, Levere RD. Hemin control of heme biosynthesis and catabolism in a human leukemia cell line. Blood. 1980; 56: 567-570. 62.Shibahara S, Yoshida T, Kikuchi G. Induction of heme oxygenase by hemin in cultured pig alveolar macrophages. Arch Biochem Biophys. 1978; 188: 243-250. 34 From bloodjournal.hematologylibrary.org by guest on June 3, 2013. For personal use only. 63.Poss KD, Tonegawa S. Heme oxygenase 1 is required for mammalian iron reutilization. Proc Natl Acad Sci U S A. 1997; 94: 10919-10924. 64.Pantopoulos K, Hentze MW. Rapid responses to oxidative stress mediated by iron regulatory protein. Embo J. 1995; 14: 2917-2924. 65.Keyse SM, Tyrrell RM. Heme oxygenase is the major 32-kDa stress protein induced in human skin fibroblasts by UVA radiation, hydrogen peroxide, and sodium arsenite. Proc Natl Acad Sci U S A. 1989; 86: 99-103. 66.Kim S, Ponka P. Control of transferrin receptor expression via nitric oxide-mediated modulation of iron-regulatory protein 2. J Biol Chem. 1999; 274: 33035-33042. 67.Corsi B, Levi S, Cozzi A, et al. Overexpression of the hereditary hemochromatosis protein, HFE, in HeLa cells induces and iron-deficient phenotype. FEBS Lett. 1999; 460: 149-152. 68.Feeney GP, Worwood M. The effects of wild-type and mutant HFE expression upon cellular iron uptake in transfected human embryonic kidney cells. Biochim Biophys Acta. 2001; 1538: 242-251. 35 From bloodjournal.hematologylibrary.org by guest on June 3, 2013. For personal use only. 69.Roy CN, Blemings KP, Deck KM, et al. Increased IRP1 and IRP2 RNA binding activity accompanies a reduction of the labile iron pool in HFE-expressing cells. J Cell Physiol. 2002; 190: 218-226. 70.Salter-Cid L, Brunmark A, Li Y, et al. Transferrin receptor is negatively modulated by the hemochromatosis protein HFE: implications for cellular iron homeostasis. Proc Natl Acad Sci U S A. 1999; 96: 5434-5439. 71.Muckenthaler M, Gray NK, Hentze MW. IRP-1 binding to ferritin mRNA prevents the recruitment of the small ribosomal subunit by the cap-binding complex eIF4F. Mol Cell. 1998; 2: 383-388. 72.Abboud S, Haile DJ. A novel mammalian iron-regulated protein involved in intracellular iron metabolism. J Biol Chem. 2000; 275: 19906-19912. 73.Donovan A, Brownlie A, Zhou Y, et al. Positional cloning of zebrafish ferroportin1 identifies a conserved vertebrate iron exporter. Nature. 2000; 403: 776-781. 74.McKie AT, Marciani P, Rolfs A, et al. A novel duodenal iron-regulated transporter, IREG1, implicated in the basolateral transfer of iron to the circulation. Mol Cell. 2000; 5: 299-309. 36 From bloodjournal.hematologylibrary.org by guest on June 3, 2013. For personal use only. 75.Knight SA, Sepuri NB, Pain D, Dancis A. Mt-Hsp70 homolog, Ssc2p, required for maturation of yeast frataxin and mitochondrial iron homeostasis. J Biol Chem. 1998; 273: 18389-18393. 76.Breuer W, Epsztejn S, Cabantchik ZI. Iron acquired from transferrin by K562 cells is delivered into a cytoplasmic pool of chelatable iron(II). J Biol Chem. 1995; 270: 2420924215. 77.Crichton RR, Ward RJ. Iron species in iron homeostasis and toxicity. Analyst. 1995; 120: 693-697. 78.Jacobs A. Low molecular weight intracellular iron transport compounds. Blood. 1977; 50: 433-439. 37 From bloodjournal.hematologylibrary.org by guest on June 3, 2013. For personal use only. Figure 1 A Hsp105 GAPDH Hsp70D HO-1 Hsp70D TfR-1 DMT-1 GAPDH actin TfR-1 HO-1 L-Fer H-Fer TfR-1 38 Figure 1 Desferrioxamine (cy3) 4x 2x H no change HO-1 D H/D 10 hsp70D 3.5 hsp105 2.7 c-myc 2.1 2x TfR-1 4x L-Fer c-jun Mt-2 lysyl oxidase L-Fer Hif-1 DMT-1 TfR 0.25 Mt-2 0.6 actin Gas-1 Gas-3 Hsp105 HO-1 mHsp70 8x c-myc Hsp70D Background cut-off Hemin (cy5) 39 1.7 0.9 From bloodjournal.hematologylibrary.org by guest on June 3, 2013. For personal use only. B 8x From bloodjournal.hematologylibrary.org by guest on June 3, 2013. For personal use only. Figure 1 C expression increase in iron loaded cells (H) H/D HO-1 10.3 Hsp70D Hsp105 mHsp70 c-myc L-Fer Gas-1 Gas-3 10.3 ±1.5 6.4 ±4.3 5.4 2.0 2.6 2.3 1.7 1.8 expression increase in iron deficient cells (D) TfR-1 5.0 DMT-1 2.0 c-jun 2.7 Mt-2 2.6 lysyl oxidase 2.4 1.5 Hif-1 D H D H/D HO-1 10 hsp70D 3.5 hsp105 2.7 c-myc 2.1 L-Fer 1.7 TfR 0.25 Mt-2 0.6 actin 0.9 6.4 ±2.5 5.4 2.0 2.6 2.3 1.7 1.8 ±0.2 ±0.3 ±0.2 ±0.2 ±0.2 H/D 0.2 0.5 0.4 0.4 0.4 0.7 ±2.2 ±0.4 ±0.4 ±0.6 ±0.4 ±0.2 40 From bloodjournal.hematologylibrary.org by guest on June 3, 2013. For personal use only. Figure 2 A Expression increase Hemin 8.0 ±0.2 3.0 ±0.5 1.8 ±0.1 1.8 ±0.1 1.7 ±0.1 2.0 ±0.2 1.7 ±0.1 HO-1 Hsp70D mHsp70 L-Fer Gas-3 Hsp105 Gas-1 FAC 2.0 ±0.2 1.6 ±0.1 1.4 ±0.05 1.5 ±0.1 1.5 ±0.1 - Expression decrease Hemin 3.0 ±1.2 2.3 ±0.3 2.0 ±0.3 2.4 ±0.4 2.0 ±0.2 TfR-1 Mt-2 c-jun lysyl oxidase Hif-1 FAC 2.0 ±0.5 1.5 ±0.2 - B Hif-1 FAC Lysyl oxidase Hemin Mt-2 c-jun TfR Gas 1 Gas 3 L-Fer mHsp70 Hsp105 Hsp70D HO-1 -4 -3 -2 41 -1 0 1 2 3 4 8 From bloodjournal.hematologylibrary.org by guest on June 3, 2013. For personal use only. Figure 3 A expression increase in H 2O2 - treated HeLa cells HO-1 TfR-1 B 2.1 ±0.1 2.0 ±0.1 expression increase in SNP - treated HeLa cells HO-1 prion Mt-1 Mt-2 4.5 2.1 1.9 1.8 ±0.5 ±0.5 ±0.2 ±0.2 expression decrease in SNP - treated HeLa cells TfR-1 DMT-1 Hsp70D hnrnpJKTP 5.0 2.0 1.9 1.8 ±2.2 ±0.3 ±0.2 ±0.2 42 From bloodjournal.hematologylibrary.org by guest on June 3, 2013. For personal use only. Figure 4 Expression increase A HFE TfR-1 c-jun lysyl oxidase Mt-2 D HFE 3.0 ±0.5 5.0 ±2 5.0 ±3 20.0 ±10 2.1 ±0.3 1.9 ±0.4 1.8 ±0.3 1.6 ±0.2 B -HFE +HFE TfR-1 -HFE/+HFE 0.6 Hsp70D 4 Hsp105 2.7 L-Fer 1.7 Gas-3 1.9 actin 1.1 Expression decrease D HO-1 Hsp70D Hsp105 mHsp70 L-Fer Gas-3 c-myc Gas-1 1.8 4.0 1.7 1.5 1.6 1.7 2.0 1.7 HFE 1.9 3.4 2.0 1.7 2.3 2.2 ±0.2 ±2 ±0.1 ±0.05 ±0.1 ±0.1 ±0.4 ±1.5 ±0.3 ±0.1 ±0.8 ±0.4 ±0.3 ±0.1 C myc HFE expression gas- 3 D treatment gas-1 L-Fer mHsp70 Hsp105 Hsp70D Ho-1 lysyl oxidase c-jun TfR-1 HFE -5 -4 -3 -2 43 -1 0 1 2 3 4 5 20 From bloodjournal.hematologylibrary.org by guest on June 3, 2013. For personal use only. Figure 5 Desferrioxamine Hemin actin L-Fer 1 2 Polysomes B 3 4 5 6 80S and mRNP Northern Desferrioxamine actin L-Fer 75% 9% 25% 91% 1 3 2 4 Polysomes 6 80S and mRNP Hemin 66% 46% 33% 54% mRNP/PS actin 0.3 0.5 mRNP/PS L-Fer 10 1.2 mRNP/PS mRNP/PS L-Fer 13 2 H-Fer 7 1.5 actin 0.6 0.5 gapdh 1 0.7 “I ronChip” 44 5 From bloodjournal.hematologylibrary.org by guest on June 3, 2013. For personal use only. Table 1 treatment gene name D Hfe FAC HO-1 Hsp70D mHsp70 L-fer Gas-3 TfR-1 Hsp105 M t-2 c-jun lysyl oxidase c-myc Gas-1 DM T-1 Hif-1 prion M t-1 hnrnpJKTP 45 Hemin SNP H 2O2