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Prepublished online as a Blood First Edition Paper on September 26, 2002; DOI 10.1182/blood-2002-07-2140.
RED CELLS
From the European Molecular Biology Laboratory,
Heidelberg; Intervet International, Schwabenheim; Department of
Medicine, University of Heidelberg; and the Department of Biocomputing,
Krebsforschungszentrum, Heidelberg, Germany.
Specialized cDNA-based microarrays (IronChips) 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 (eg, oxidative stress, nitric
oxide [NO] metabolism, or copper metabolism), and were
immobilized on glass slides. HeLa cells were treated with iron
donors or iron chelators, or were subjected to oxidative stress
(H2O2) or NO (sodium nitroprusside). 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
signal-specific 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
iron-regulatory protein (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 the
discrimination 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.
(Blood. 2003;101:3690-3698) 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 noniron signals
such as nitric oxide (NO) and oxidative
stress.5,6
Iron homeostasis is regulated at the systemic and the cellular level.
The expression of central proteins involved in iron uptake and
transport, iron storage, and iron usage is controlled by the
iron-responsive element (IRE)/iron-regulatory protein (IRP) regulatory system. IREs are RNA elements that function as
binding sites for IRP-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, for example, the iron-storage proteins H- and
L-ferritin, the erythroid 5-aminolevulinate synthase (eALAS), and
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 Muckenthaler and Hentze,13 Cairo and
Pietrangelo,14 and Eisenstein15).
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 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 posttranscriptional 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- 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 a major histocompatibility complex
(MHC) class 1-like protein37 that forms a
heterodimer with Gene-expression profiling using DNA microarrays has allowed
gene-expression analyses to be broadened from the study of single genes
to the investigation of 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 sodium nitroprusside (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, for both IRP and
non-IRP target genes. This finding suggests that HFE expression
directly targets the regulatory iron pool(s) of the transfected cells.
Selection of cDNA clones
For the IronChip (version 2.0)
(http://www.embl-heidelberg.de/ExternalInfo/hentze/suppinfo.html;
accessed January 16, 2003), 113 human EST clones that were
sequence-verified from both ends were chosen. The ESTs were selected to
contain the 3' end of a cDNA (ie, the polyadenylation signal) and to
extend for at least 300 bp toward the 5' end. The clone-finder
software, developed by the HUSAR Biocomputing Service Group at the
German Cancer Research Center (Heidelberg, Germany)
(http://genome.dkfz-heidelberg.de; accessed January 16, 2003)
facilitated the selection. The clones were purchased from the
German Resource Center and Primary Database (RZPD; Berlin and Heidelberg).
Preparation of the IronChip microarray platform
Synthesis of fluorescent cDNA probes Fluorescent cDNA probes were synthesized from 5 µg total RNA by means of a linear mRNA amplification protocol, exactly as described in http://cmgm.stanford.edu/pbrown/protocols/ampprotocol_3.html (accessed January 16, 2003). Subsequently, 3 µg T7 RNA polymerase-amplified antisense RNA was subjected to a direct labeling reaction by incorporation of cyanin 3 (Cy3) and Cy5 fluorescent dyes (Cy3 or Cy5) with the use of random primers (http://cmgm.stanford.edu/pbrown/protocols/4_human_RNA.html; accessed January 16, 2003).53At least 2 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 2 different dyes. Genes were scored as differentially expressed only if they displayed a consistent regulatory pattern in such dye-switch experiments. Microarray analysis The microarrays were immersed at 42°C in 6 × standard saline citrate (SSC)/0.5% sodium dodecyl sulfate (SDS)/1% bovine serum albumin (BSA) for 40 minutes and subsequently washed extensively with ddH2O at room temperature. Prior to hybridization, the spotted polymerase chain reaction (PCR) products were denatured by immersing the slides at 95°C in double-distilled (dd) H2O for 2 minutes. Excess of liquid was removed from the slides by centrifuging them briefly at 715g in a microtiter plate centrifuge (Z320; Hermle, Wehingen, Germany). Prior to hybridization, the purified Cy3- and Cy5-labeled cDNAs were mixed; 5 µg polydeoxyadenosine (poly(dA)) and 1 µg human Cot1 DNA (both Gibco Invitrogen, Carlsbad, CA) were added and subsequently evaporated in a vacuum Concentrator 5301 (Eppendorf, Hamburg, Germany) at 60°C. The resulting pellet was dissolved in 12 µL hybridization buffer (50% formamide/6 × SSC/0.5% SDS/5 × Denhardt) and denatured by incubating at 95°C for 2 minutes. The probe was then transferred onto the array under a 24 × 24 mm coverslip and incubated in a humid chamber (GeneMachines, San Carlos, CA) containing 2 × SSC drops for providing humidity. Hybridization was performed for 12 to 16 hours in a 42°C water bath (GFL, Burgwedel, Germany).After hybridization, the microarrays were washed in 0.1 × SSC/0.1% SDS for 10 minutes and twice with 0.1 × SSC for 5 minutes (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 715g in a microtiter plate centrifuge (Z320, Hermle). 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). 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 by means of the ChipSkipper microarray data evaluation software (http://pc-ansorge11.embl-heidelberg.de/ chipskipper; accessed January 16, 2003). 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, Redmond, WA). At least 2 independent cell culture experiments were performed for each experimental condition tested and analyzed on the IronChip (version 2.0). For the 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 the ratios of those different clones was calculated. The standard deviation for each resulting ratio was determined. Genes listed in Table 6 represent those that have been scored as differentially expressed in all the experiments performed for a specific treatment. Genes are scored as differentially expressed if the calculated ratios exceed the ratio cutoff value, defined by the use 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 100 µM hemin, 100 µM ferric ammonium citrate, 100 µM desferrioxamine, 100 µM H2O2, and 100 µM SNP were performed as described previously.27 All treatments were performed for 8 hours. The establishment and maintenance of the HFE-overexpressing cell line as well as the experimental conditions of HFE overexpression are described in Riedel et al.47 Total RNA from HeLa cells was extracted by means of RNAclean (Hybaid-AGS, Heidelberg, Germany) according to the manufacturer's instructions.For Northern analysis, 10 µg total RNA was 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, Piscataway, NJ). Sucrose gradient analysis 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 White and Munro.56
Validation of the human IronChip We first established a cDNA microarray platform that represents a selection of human genes that are directly involved in iron metabolism or that 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 For version 2.0 of the IronChip, 113 different human genes represented by up to 3 independent cDNA clones were selected. The names of these genes and their GenBank accession numbers are shown at http://www.embl-heidelberg.de/ExternalInfo/hentze/suppinfo.html. In addition, GenBank accession numbers are included in the text for all IronChip genes mentioned.To assess whether the IronChip reflects changes in mRNA levels in
response to iron perturbations, HeLa cells were either iron loaded by
treatment with 100 µM hemin (H) for 8 hours, or made iron deficient
by incubation with 100 µM desferrioxamine (D) for 8 hours. Total RNA
was purified from the treated cells, labeled with Cy3 (D) and Cy5 (H),
or vice versa ("Materials and methods"), and
analyzed on the IronChip. The results of
these experiments are shown in Figure 1 and Table
1. 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 2-fold
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, which 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 Housekeeping genes, like glyceraldehyde phosphate dehydrogenase (GAPDH) (M33197) or
In addition to those genes that are directly involved in iron
metabolism, we found some additional genes to be regulated. In
iron-replete cells, 3 members of the heat-shock protein (hsp) family
(hsp70D [M11717], hsp105 Gene-expression profiles derived from hemin- and ferric ammonium citrate (FAC)-treated HeLa cells We next assessed HeLa cells that were treated with 2 different sources of iron, hemin (ferric protoporphyrin IX) or ferric ammonium citrate (FAC), to address 2 questions: first, whether the iron-loaded state resulting from both treatments elicited a common pattern in the expression profiles; second, whether these 2 similar treatments could be discriminated by diagnostic features of the gene-response patterns.Subconfluent HeLa cells were treated with either 100 µM hemin
or 100 µM FAC for 8 hours. Untreated HeLa cells were used as a
control for both. As can be seen in Figure
2 and Table
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, Gas-3, TfR-1, and
Mt-2. The increased expression of the first 5 and the
decreased expression of the last 2 genes appear to define a
common denominator that is the hallmark of 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 used as the minimum defining cutoff
between regulated and unregulated genes; "Materials and methods")
of hsp105
Gene-expression profiles of H2O2- and sodium nitroprusside (SNP)-treated HeLa cells H2O2 treatment and iron deficiency both activate IRP-122-28 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 100 µM H2O2 for 8 hours, and total RNA was subsequently analyzed on the IronChip in comparison with total RNA from untreated control cells. H2O2 treatment induced increased HO-1 and TfR-1 mRNA levels (Table 3). The induction of HO-1 by H2O2 has been reported previously.65 By contrast, we observe neither the regulation of the IRE-containing DMT1/DCT1/Nramp2 mRNA nor any regulation of those genes that are seen regulated in iron-deficient HeLa cells (Table 4). 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, nitric oxide also 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 100 µM SNP for 8 hours; total
RNA was extracted and analyzed on the IronChip in comparison with an
untreated control sample. As expected, SNP treatment reduces TfR mRNA
levels (Table 5). In addition, the mRNA
levels of the IRE-containing splice variant DMT1/DCT1/Nramp2 are also
reduced. The approximately 2-fold increase of DMT1/DCT1/Nramp2 mRNA in iron deficiency (when compared with hemin-treated cells; Figure 1 and
Table 1) and its reduced expression in response to SNP are consistent
with an IRP-mediated regulatory mechanism of DCT1/DMT1/Nramp2 mRNA
stability. In contrast to iron-manipulated HeLa cells, hsp70D is
coregulated with TfR-1 and DCT-1 in SNP-treated cells. SNP treatment
strongly induces HO-1 and affects the expression of the
metallothioneins 1 and 2 (Table 5). 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 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 more globally record the responses of transfected HeLa cells to induced HFE expression 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 (Table
4). 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-ferritin (L-fer), and
Gas-3 decrease. These data were confirmed by Northern analysis (Figure
3A). Doxycyclin treatment of
nontransfected HeLa cells did not affect the regulation of IronChip
genes (data not shown). When HeLa cells were treated with 100 µM
desferrioxamine, TfR-1, c-jun, and lysyl oxidase mRNA levels increased.
Decreased mRNA levels were found for HO-1, Hsp70D, Hsp105a, mHsp70,
L-fer, Gas-1, Gas-3, and c-myc (Table 4).
These data sets reveal striking similarities between the gene
responses elicited by HFE expression and desferrioxamine
treatment. This conclusion applies both to IRP target genes
and non-IRP target genes (Figure 3B; Table 4). Both gene-response
patterns differ significantly from those elicited by, for example, SNP
and H2O2 treatment (Figure 3; Table 6). 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 ("Materials and methods"). Six
fractions were prepared from each sample; total RNA was extracted and
initially analyzed by Northern blotting. As shown in Figure 4, 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 messenger ribonucleic proteins (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.
For microarray analyses, we pooled the total RNA derived from the polysomal fractions 1 through 3 and the total RNA derived from the monosomal/mRNP fractions 4 and 5. For each condition (hemin and desferrioxamine treatment), the polysomal and monosomal/mRNP fractions were labeled with different fluorescent dyes and hybridized to the IronChip. Similarly to the Northern blots, the data (Figure 4B) clearly reveal the translational regulation of L-ferritin mRNA and the lack of actin mRNA regulation. Similarly to L-ferritin, the IronChip also reflects the translational control of the H-ferritin mRNA in response to iron perturbation (Figure 4). 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 ironregulated transporter (IREG)-1/ferroportin/metal transporter protein-1 (MTP-1) is expressed mainly 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.
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). With this approach, novel insights into human iron metabolism were obtained. 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. 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 6 provides a synopsis of 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 (Figures 1C and 3A), which in addition ascertained a surprisingly good performance of the microarray platform in yielding quantitatively accurate data.Heme oxygenase 1 (HO-1) emerges as the most strongly responsive gene in our data set. HO-1 mRNA levels decrease in iron-deficient and in HFE-expressing cells, and increase in response to iron loading as well as to SNP and H2O2 exposure (Table 6). 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 might have responded if a different cell line had been tested. Only 2 genes displayed H2O2 regulation under our experimental conditions (Table 6). 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 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 human 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 receptor43,46,47,67-70 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 yield nearly identical
responses of the 113 genes represented on the IronChip. This allows the
conclusion that not only does HFE expression in this experimental
system trigger an iron-deficiency response by the IRE/IRP network, but that 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
(eg, c-jun, Gas-3) and stress-response genes (eg, HO-1, Hsp70D,
Hsp105 The similarity between the genetic responses to iron overload by hemin and ferric ammonium citrate (FAC) was predicted and has been confirmed with the IronChip (Figure 2; Table 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, Gas-3, TfR1, and Mt-2 is diagnostic for hemin-induced versus FAC-induced iron overload. More generally, this analysis provides an example for the possibility of discriminating between 2 related stimuli by IronChip analysis. The IronChip provides a versatile tool for the analysis of iron metabolism As illustrated, the human IronChip was validated as a reliable assay system to identify the responses of more than 100 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 (Figure 4). This is particularly pertinent for the study of mammalian iron metabolism.13Compared with far more comprehensive cDNA or oligonucleotide-based microarrays, the IronChip offers only limited chances to identify "new genes" that are regulated by 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 as is being used for updated versions. On the other hand, we believe that both the 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. Owing 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 3 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 the ability 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 model systems for human diseases of iron metabolism and to integrate findings from animal models into our understanding of human iron physiology and pathophysiology.
We thank the Resource Center and Primary Database (RZPD) for the supply of IMAGE clones.
Submitted August 15, 2002; accepted August 15, 2002.
Prepublished online as Blood First Edition Paper, September 26, 2002; DOI 10.1182/blood-2002-07-2140.
Supported by funds from the Gottfried Wilhelm Leibniz Prize to M.W.H., which were used to establish the IronChip.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Matthias W. Hentze, European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117 Heidelberg, Germany; e-mail: hentze{at}embl.de.
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Top
Abstract
Introduction
(Hif-1
(IL-1
1.4-fold) are represented in green.
Housekeeping genes, like GAPDH and actin, are represented in yellow,
and negative controls are shown in blue. Positive spike-in
controls
