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RED CELLS
Epolones induce erythropoietin expression via hypoxia-inducible
factor-1␣ activation
Roger M. Wanner, Patrick Spielmann, Deborah M. Stroka, Gieri Camenisch, Isabelle Camenisch, Annette Scheid, David R. Houck,
Christian Bauer, Max Gassmann, and Roland H. Wenger
Induction of erythropoietin (Epo) expression under hypoxic conditions is mediated by the heterodimeric hypoxia-inducible factor (HIF)-1. Following binding to
the 3ⴕ hypoxia-response element (HRE) of
the Epo gene, HIF-1 markedly enhances
Epo transcription. To facilitate the search
for HIF-1 (ant)agonists, a hypoxiareporter cell line (termed HRCHO5) was
constructed containing a stably integrated luciferase gene under the control
of triplicated heterologous HREs. Among
various agents tested, we identified a
class of substances called epolones,
which induced HRE-dependent reporter
gene activity in HRCHO5 cells. Epolones
are fungal products known to induce Epo
expression in hepatoma cells. We found
that epolones (optimal concentration 4-8
␮mol/L) potently induce HIF-1␣ protein
accumulation and nuclear translocation
as well as HIF-1 DNA binding and reporter
gene transactivation. Interestingly, the activity of a compound related to the fungal
epolones, ciclopirox olamine (CPX), was
blocked after addition of ferrous iron.
This suggests that CPX might interfere
with the putative heme oxygen sensor, as
has been proposed for the iron chelator
deferoxamine mesylate (DFX). However,
about 10-fold higher concentrations of
DFX (50-100 ␮mol/L) than CPX were re-
quired to maximally induce reporter gene
activity in HRCHO5 cells. Moreover, structural, functional, and spectrophotometric
data imply a chelator:iron stoichiometry
of 1:1 for DFX but 3:1 for CPX. Because
the iron concentration in the cell culture
medium was determined to be 16 ␮mol/L,
DFX but not CPX function can be explained by complete chelation of medium
iron. These results suggest that the lipophilic epolones might induce HIF-1␣ by
intracellular iron chelation. (Blood. 2000;
96:1558-1565)
© 2000 by The American Society of Hematology
Introduction
The glycoprotein hormone erythropoietin (Epo), produced by the
embryonic liver and the adult kidney, is the main stimulator of
erythropoiesis.1 Recombinant Epo is widely used to treat patients
suffering from anemia. However, recombinant Epo is expensive
and must be administered intravenously or subcutaneously. Thus,
an orally active, small molecular weight compound that induces
endogenous Epo production would be an alternative for the
treatment of anemia not caused by deficient renal Epo production.
Therefore, fungal products were screened for their ability to induce
a reporter gene under the control of 6-kilobase (kb) 5⬘ and 0.3-kb 3⬘
flanking sequences derived from the Epo gene.2-4 Several compounds (the 3 sesquiterpene tropolones: pycnidione, epolone A and
epolone B, and 8-methyl-pyridoxatin) were isolated, which induced reporter gene expression as well as Epo production in human
Hep3B hepatoma cells.2,3 In addition, the related commercially
available pyridone, ciclopirox olamine (CPX), was shown to have a
similar function.2 Hereinafter, we refer to this class of substances,
capable of inducing Epo expression, as “epolones.” The mechanism of Epo induction via epolones and the involved cis-regulatory
elements, however, remained unidentified.
The most powerful inducer of Epo expression is hypoxia (a
deficiency in oxygen supply not matching oxygen consumption).
The cellular oxygen concentration is measured by a putative heme
oxygen sensor.5 Hypoxia results in the activation of hypoxiainducible factor (HIF)-1, a heterodimeric transcription factor
containing an oxygen-labile ␣ subunit and a constitutively expressed ␤ subunit previously known as aryl hydrocarbon receptor
nuclear translocator (ARNT).6 Upon exposure to hypoxia, HIF-1␣
becomes protected from proteolytic degradation, translocates into
the nucleus, and heterodimerizes with ARNT to form the HIF-1
complex.7-10 HIF-1 then binds to its DNA consensus binding site
(HBS) present within cis-regulatory hypoxia-response elements
(HREs) of oxygen-regulated genes.9 HREs confer enhanced transcription of oxygen-regulated genes by recruiting various other
transcription factors in addition to HIF-1 along with transcriptional
coactivators such as CBP/p300.7 These “oxygen(es)”9 include Epo,
vascular endothelial growth factor (VEGF), glycolytic enzymes,
and glucose transporter as well as transferrin and transferrin
receptor. Apart from hypoxia, the transition cations Co2⫹ and Ni2⫹
as well as the iron chelator deferoxamine mesylate (DFX) are
capable of inducing HIF-1␣ protein stability and hypoxiadependent gene expression, probably by replacing or removing the
central iron of the putative heme oxygen sensor.5,7,11
In this work, we established an HIF-1–dependent hypoxiareporter cell line, investigated the effects of epolones on HIF-1
From the Institute of Physiology, University of Zürich-Irchel, Zürich,
Switzerland; and OSI Pharmaceuticals, MYCOsearch, Durham, NC.
Reprints: Roland H. Wenger, Institut für Physiologie, Medizinische Universität
zu Lübeck, Ratzeburger Allee 160, D-23538 Lübeck, Germany; e-mail:
wenger@physio.mu-luebeck.de.
Submitted February 28, 2000; accepted April 21, 2000.
Supported by the Käthe Zingg-Schwichtenberg-Fonds, the Novartis Stiftung,
and the Swiss National Science Foundation (grant 31-56743.99). A.S. is a
recipient of a Deutsche Forschungsgemeinschaft fellowship. R.H.W. is a recipient
of the Sondermassnahmen des Bundes zur Förderung des akademischen
Nachwuchses.
1558
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.
© 2000 by The American Society of Hematology
BLOOD, 15 AUGUST 2000 䡠 VOLUME 96, NUMBER 4
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BLOOD, 15 AUGUST 2000 䡠 VOLUME 96, NUMBER 4
activation, and unraveled the mechanism by which epolones
activate Epo expression under normoxic conditions.
Materials and methods
Reagents
DFX, 2,2⬘-dipyridyl, CPX, neocuproine, ferrozine, luminol, and coumaric
acid were purchased from Sigma (Buchs, Switzerland) and ferrous ethylenediammonium sulfate from Fluka (Buchs, Switzerland). Pycnidione and
8-methyl-pyridoxatin were isolated as described previously.2,3 Stock solutions (50 mmol/L) of the epolones (CPX, pycnidione, and 8-methylpyridoxatin) were prepared in methanol and stored at ⫺30°C. Immediately
before use, they were diluted in water to a working concentration of
1 mmol/L.
Cell culture and transfection
Chinese hamster ovary (CHO) cells were a kind gift of Peter J. Nielsen
(Freiburg, Germany), and human hepatoma HepG2 cells were purchased
from the American Type Culture Collection (HB-8065). Both cell lines
were cultured in Dulbecco’s modified Eagle’s medium (DMEM; high
glucose, Life Technologies) supplemented with 10% heat-inactivated fetal
calf serum (FCS; Boehringer-Mannheim, Basel, Switzerland), 100-U/mL
penicillin, 100-␮g/mL streptomycin, 1 ⫻ nonessential amino acids, and
1-mmol/L Na-pyruvate (all purchased from Life Technologies, Basel,
Switzerland) in a humidified atmosphere containing 5% CO2 at 37°C.
Oxygen partial pressures in the incubator (Forma Scientific, Illkirch,
France) were either 140 mmHg (20% O2 vol/vol, normoxia) or 7 mmHg
(1% O2 vol/vol, hypoxia). For transfection, 0.5 ⫻ 106 cells in 350-␮L
medium without FCS were mixed with 50 ␮g of DNA in 50 ␮L of
10-mmol/L Tris-HCl (pH 7.4), and 1-mmol/L ethylenediaminetetraacetic
acid and electroporated at 250 V and 960 ␮F (GenePulser, Bio-Rad,
Glattbrugg, Switzerland).
Hypoxia-reporter assays
A firefly luciferase reporter gene plasmid (pH3SVL) containing a total of 6
HBSs derived from the transferrin HRE12 was constructed by inserting 2
copies of the oligonucleotide TfHBSww into the SmaI site of the plasmid
pGLTfHBSww.12 For stable transfection, pH3SVL was linearized with
XmnI, mixed with the EcoRI-linearized neomycin expression vector
pSV2neo at a molar ratio of 100:1, and coelectroporated into CHO cells.
Following limited dilution and selection in 2-mg/mL G418 (Alexis,
Läufelfingen, Switzerland), a hypoxia-reporter cell line (termed HRCHO5)
was chosen based on the efficiency of hypoxic reporter gene induction. For
transient transfection assays, pGLHIF1.3 containing 3 copies of the HBS
derived from the Epo 3⬘ HRE13 was coelectroporated into HepG2 cells
together with the ␤-galactosidase reference vector pCMVlacZ.12 The cells
were split and incubated for 43 hours under normoxic or hypoxic
conditions. Following stimulation, stably transfected HRCHO5 cells and
transiently transfected HepG2 cells were lysed in reporter lysis buffer
(Promega), and luciferase and ␤-galactosidase activities were determined
according to the manufacturer’s instructions (Promega, Catalys, Wallisellen, Switzerland) using a Lumat LB9501 luminometer (EG&G Berthold,
Regensdorf, Switzerland) and a DigiScan 96-well plate photometer (ASYS,
BioBlock, Illkirch, France), respectively. Differences in the transfection
efficiency and extract preparation were corrected by normalization to the
corresponding protein contents (Bradford assay, Bio-Rad) or ␤-galactosidase activities.
Immunoblot and immunofluorescence analysis
Following stimulation, cells were harvested and nuclear extracts were
prepared as described previously,13 except that the cells were lysed with
0.02% Nonidet P-40 instead of dounce homogenization. For immunoblot
assays, aliquots (30 ␮g) of nuclear extracts were fractionated by 7.5%
sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrotrans-
EPOLONES ACTIVATE HIF-1␣
1559
ferred to a nitrocellulose membrane (Schleicher & Schuell, Riehen,
Switzerland). Staining with PonceauS (Sigma) confirmed equal loading and
blotting efficiency. After blocking nonspecific binding sites with 4%
defatted milk powder in phosphate-buffered saline (PBS), the blot was
probed with the affinity-purified anti–HIF-1␣ monoclonal antibody mgc3
described previously14 (Affinity BioReagents, Lausen, Switzerland), followed by a horseradish peroxidase–coupled secondary goat antimouse
antibody (Pierce, Socochim, Lausanne, Switzerland). Chemiluminescence
detection was performed by incubation of the membrane with 100-mmol/L
Tris-HCl (pH 8.5), 2.65-mmol/L H2O2, 0.45-mmol/L luminol, and 0.625mmol/L coumaric acid for 1 minute, followed by exposure to x-ray films
(SuperRX, Fuji, Dielsdorf, Switzerland). For immunofluorescence analysis,
adherent cells were fixed with freshly prepared 4% paraformaldehyde in
PBS (pH 7.4) for 10 minutes, washed with PBS, permeabilized with 0.5%
Triton X-100 for 5 minutes, and rinsed again with PBS. After blocking
nonspecific binding sites with 10% FCS in PBS for 30 minutes, the cells
were incubated overnight with the anti–HIF-1␣ antibody mgc3 diluted 1:10
with 3% BSA in PBS, followed by a fluorescein isothiocyanate–coupled
secondary donkey antimouse antibody diluted 1:100 with 3% BSA in PBS
(Jackson, Milan Analytica, La Roche, Switzerland). After extensive washings in PBS and mounting in Mowiol (Calbiochem, Stehelin, Basel,
Switzerland), the cells were analyzed by fluorescence microscopy.
Electrophoretic mobility shift assay
Electrophoretic mobility shift assays (EMSAs) were carried out as described previously.15 A double-stranded, HBS-containing oligonucleotide
derived from the Epo 3⬘ enhancer was used as probe. For supershift
analysis, the anti–HIF-1␣ monoclonal antibody mgc3 was added to the
completed DNA-protein binding reaction mixture and incubated for 16
hours at 4°C prior to loading.
Iron determinations
Iron concentrations were determined by a colorimetric assay according to
Fish.16 Briefly, iron was released from 100-␮L samples by treatment with
50 ␮L of 142-mmol/L KMnO4 and 600-mmol/L HCl at 60°C for 2 hours.
Thereafter, 10 ␮L of 5-mol/L ammonium acetate, 2-mol/L ascorbic acid,
13.1-mmol/L neocuproine, and 6.5-mmol/L ferrozine were added and
incubated at room temperature for 30 minutes. Iron was determined by
measuring the absorption at 562 nm in a DU-65 spectrophotometer
(Beckmann). Iron standards were prepared by dissolving ferrous ethylenediammonium sulfate in 10-mmol/L HCl. The detection limit of this assay was
2 ng of iron, and the standard curve was linear up to 800 ng of iron. It has
been validated by demonstrating a 2-fold molar ratio of iron to protein in
10-␮g samples of iron-saturated purified transferrin (Life Technologies).
Results
Generation of the HRCHO5 hypoxia-reporter cell line
To facilitate the screening for novel agonists/antagonists of the
oxygen-regulated signaling pathway, CHO cells were stably transfected with the hypoxia-dependent reporter gene shown in Figure
1A. This construct contains the firefly luciferase cDNA under the
control of the SV40 promoter and 3 HREs derived from the
hypoxia-responsive transferrin 5⬘ enhancer.12 Of note, the transferrin HRE contains 2 HBSs and the whole construct, hence, a total
of 6 HBSs. One CHO clone (designated HRCHO5) was selected
based on its high hypoxic inducibility of luciferase activity.
Exposure of HRCHO5 cells to hypoxic conditions (1% oxygen) for
18 hours led to a 21.2 ⫾ 4.2-fold induction (mean ⫾ SD, n ⫽ 9) of
luciferase activity compared with normoxic (20% oxygen) control
cells (Figure 1B). The transition cations Co2⫹ and Ni2⫹ dosedependently induced luciferase activity under normoxic conditions,
approximately reaching the hypoxic values at concentrations of 50
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1560
WANNER et al
BLOOD, 15 AUGUST 2000 䡠 VOLUME 96, NUMBER 4
we reasoned that epolones could activate Epo transcription via
an HBS, which is also present in the hypoxia-reporter cell line
HRCHO5. Indeed, treatment of HRCHO5 cells with the epolones CPX, pycnidione, and 8-methyl-pyridoxatin dose-dependently induced luciferase expression under normoxic conditions
(Figure 2, top graph). While the induction was maximal at
epolone concentrations of 4 to 8 ␮mol/L (16 ␮mol/L for CPX),
luciferase activity decreased again at higher doses. Hypoxic
exposure of HRCHO5 cells (Figure 2, bottom graph) had
additive effects, but the decrease in reporter gene activity
occurred already at lower epolone concentrations than under
normoxic conditions.
The epolone CPX activates reporter gene
expression via the HRE
We next analyzed whether epolones activated luciferase expression
specifically via the HRE or whether other cis-regulatory elements
were involved, as could be expected from the fact that hypoxia and
epolones had additive effects. Therefore, luciferase reporter gene
constructs containing 3 copies of the Epo 3⬘ HBS, either wild type
(pGLHIF1.3) or mutant (pGLHIF1mt.3),13 were transiently transfected into HepG2 cells, which were split and exposed to hypoxia
and/or CPX. As shown in Figure 3, CPX increased reporter gene
activity also in this reporter gene–cell line combination under both
normoxic and hypoxic conditions. However, the mutant HBSs
Figure 1. Activation of reporter gene expression in HRCHO5 cells by hypoxia,
transition metals, and iron chelation. (A) Reporter gene construct used to
generate the hypoxia-reporter cell line HRCHO5. TfHRE, hypoxia-response
element derived from the transferrin 5⬘ enhancer12; SV40, simian virus 40 early
promoter. (B) Luciferase activities in HRCHO5 cells following stimulation with
CoCl2, NiCl2, and DFX for 18 hours under normoxic (20% oxygen) or hypoxic (1%
oxygen) conditions. After preparation of cell extracts, determination of the
luciferase activities, and normalization to the protein contents, the results were
expressed as fold increases over the untreated normoxic controls. Means ⫾ SD of
3 independent experiments.
␮mol/L (CoCl2) and 100 ␮mol/L (NiCl2), respectively. Luciferase
activity dropped again at higher concentrations, presumably because these agents are cytotoxic. Similar results were obtained with
the iron chelator DFX, which maximally induced normoxic
luciferase activity at concentrations of 50 to 200 ␮mol/L (Figure 1B, top
graph). Under hypoxic conditions, CoCl2 and DFX additionally induced
reporter gene activity with a similar dosage dependence as found under
normoxic conditions. In contrast, NiCl2 had no additional effects under
hypoxic conditions (Figure 1B, bottom graph).
Epolones induce reporter gene activity in HRCHO5 cells
under normoxic and hypoxic conditions
Epolones have previously been identified as a family of fungal
products capable of inducing Epo expression under normoxic
conditions.2,3 Because the reporter gene used for Epo-inducing
drug screening contained the 3⬘ HRE derived from the Epo gene,
Figure 2. Activation of reporter gene expression in HRCHO5 cells by epolones.
Luciferase activities in HRCHO5 cells following stimulation with the indicated
concentrations of the 3 epolones—CPX, pycnidione, and 8-methyl-pyridoxatin—for
18 hours under normoxic (20% oxygen) or hypoxic (1% oxygen) conditions.
Luciferase activities were determined as described in Figure 1. Means ⫾ SD of 3
independent experiments.
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BLOOD, 15 AUGUST 2000 䡠 VOLUME 96, NUMBER 4
EPOLONES ACTIVATE HIF-1␣
1561
moxic HepG2 cells to the same extent as CoCl2. Hypoxic
conditions (Figure 4, right part) also induced HIF-1␣ protein,
which could be further enhanced by adding 8-methyl-pyridoxatin
or CPX but not pycnidione or CoCl2. Next, as shown by immunofluorescence, hypoxia as well as the epolone CPX induced nuclear
accumulation of HIF-1␣ in HepG2 cells (Figure 5). Finally, as
shown by EMSA, CPX also activated DNA binding of HIF-1 to an
oligonucleotide probe containing an HBS derived from the Epo
HRE (Figure 6). This induction was slightly more pronounced than
with hypoxia or DFX or the combination of hypoxia with CPX.
Supershift analysis using the monoclonal anti–HIF-1␣ antibody
mgc314 confirmed that (most of) the HIF complexes present in
HepG2 nuclear extracts contained the HIF-1␣ subunit.
Figure 3. Activation of reporter gene expression in transiently transfected
HepG2 hepatoma cells by CPX. HepG2 cells were cotransfected with the indicated
luciferase reporter gene constructs together with a ␤-galactosidase control expression vector. Following splitting and stimulation with hypoxia (1% oxygen) and/or CPX
(8 ␮mol/L) for 43 hours, reporter gene activities were determined and expressed as a
ratio between luciferase and ␤-galactosidase activities. The luciferase constructs
contained 3 wild-type HBSs (pGLHIF1.3) or 3 mutant HBSs (pGLHIF1mt.3) as
described previously.13 The empty parental vector pGL3 promoter was included as
control. Means ⫾ SD of 3 independent experiments.
conferred neither CPX nor hypoxic induction of reporter gene
expression, suggesting that both stimuli activate reporter gene
expression via similar mechanisms.
Epolones induce HIF-1␣ protein stability, nuclear
translocation, and DNA binding activity
The binding of HIF-1 to an HBS is obligatory for the activation of
an HRE and subsequent gene expression. The sequential steps in
the formation of the HIF-1 complex include the stabilization of its
␣ subunit followed by nuclear translocation and heterodimerization
with ARNT to form a functional DNA-binding transcription factor
complex. We thus investigated whether this pathway could be
mimicked by epolones.
First, HIF-1␣ protein levels were determined by immunoblotting in HepG2 cells. As shown in the left part of Figure 4, all 3
epolones efficiently induced HIF-1␣ protein expression in nor-
Figure 4. Immunoblot analysis of HIF-1␣ protein expression in HepG2 cells.
HepG2 cells were treated with the epolones pycnidione, 8-methyl-pyridoxatin, and
CPX (8 ␮mol/L) as well as with CoCl2 (100 ␮mol/L) for 4 hours under normoxic (20%
oxygen) or hypoxic (1% oxygen) conditions. HIF-1␣ protein was detected by
immunoblotting of nuclear extracts using the monoclonal anti–HIF-1␣ antibody
mgc3.14
Iron blocks DFX- and CPX-mediated induction
of HIF-1-dependent gene activation
Having established that epolone-dependent reporter gene activation followed HIF-1␣ induction, we addressed the question of how
epolones might induce HIF-1␣. Based on functional (Figures 1 and
2) and structural similarities between DFX and CPX, we followed
the hypothesis that CPX also might act as an iron chelator.
Feroxamine contains 1 atom of ferrous iron in the center of a
hexadentate cluster formed by 3 hydroxamic acid groups.17 The
epolone CPX contains a single hydroxamic acid group, suggesting
a bidentate structure theoretically requiring 3 mol of CPX to
chelate 1 mol of iron. We thus titered the concentration of iron
necessary to block DFX- and CPX-induced reporter gene activation in HRCHO5 cells. As shown in Figure 7A, efficient inhibition
of luciferase activity under normoxic and hypoxic conditions
occurred at an iron:DFX molar ratio of 1:1, consistent with the
ability of 1 molecule of DFX to stoichiometrically chelate 1
molecule of iron. Interestingly, CPX-induced luciferase activity
was abolished at an iron:CPX ratio of 1:2 but not at 1:4, supporting
the idea that CPX functions as an iron chelator with the expected
stoichiometry of 3 mol of CPX per 1 mol of iron.
As shown in Figure 7B, the iron chelation–dependent induction
of reporter gene activity in HRCHO5 cells can also be blocked by
addition of AlCl3. However, while inhibition of DFX activity again
Figure 5. Immunofluorescence analysis of HIF-1␣ expression in HepG2 cells.
HepG2 cells were treated with hypoxia (1% oxygen) and/or CPX (8 ␮mol/L) for 4
hours, and HIF-1␣ was detected using the monoclonal antibody mgc314 followed by a
fluorescein isothiocyanate–conjugated secondary antibody.
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WANNER et al
BLOOD, 15 AUGUST 2000 䡠 VOLUME 96, NUMBER 4
show iron-dependent (but not aluminium-dependent) absorption
maxima at 430 nm19 and 421 nm, respectively, allowing the direct
estimation of the iron chelation stoichiometry. Whereas DFX iron
saturation was found to be completed at a 1:1 molar ratio, CPX was
saturated with iron at a molar ratio of 1:3 (Figure 9B), thus
confirming the functional results obtained in the HRCHO5 cell line.
In this context, it would be important to know the iron
Figure 6. EMSA of HepG2 nuclear extracts. HepG2 cells were treated for 4 hours
with DFX (100 ␮mol/L) or CPX (8 ␮mol/L) under normoxic (20% oxygen) or hypoxic
(1% oxygen) conditions. Nuclear extracts were incubated with a radioactively labeled
oligonucleotide probe derived from the Epo 3⬘ HRE13 and separated by native
polyacrylamide gel electrophoresis. Specific HIF-1 DNA binding was confirmed by
supershift analysis using the monoclonal antibody mgc3.14
required stoichiometric concentrations of AlCl3, the activity of
CPX was already reduced by approximately 50% at a molar
aluminium:CPX ratio of 1:8. This cannot be attributed to a higher
toxicity of AlCl3 compared with FeCl2 because the actual AlCl3
concentration inhibiting CPX function was only 1 ␮mol/L, whereas
50-␮mol/L AlCl3 had no significant effect on DFX-induced reporter gene activity. Thus, these data suggest that CPX-mediated
activation of luciferase expression in HRCHO5 cells might be due
to metal chelation.
To exclude the possibility of an inhibitory function of iron
downstream of HIF-1␣ induction, we analyzed HIF-1␣ protein
expression directly by immunoblotting. As shown in Figure 8,
addition of iron inhibited HIF-1␣ induction by CPX as well as by
the established activators DFX11 and 2,2⬘-dipyridyl.18
Stoichiometry of iron chelation by DFX and CPX and its relation
to the cell culture medium iron concentration
The iron chelation stoichiometry suggested by the titration experiments shown in Figure 7 might be compromised by the (unknown)
concentration of iron in the cell culture medium. To confirm our
data, the iron saturation curves of DFX and CPX were determined
by spectrophotometry. As shown in Figure 9A, DFX and CPX
Figure 7. Iron and aluminium block the DFX- and CPX-mediated reporter gene
induction in HRCHO5 cells. The cells were stimulated with the optimal DFX (100
␮mol/L) and CPX (8 ␮mol/L) concentrations, as determined in Figures 1 and 2,
respectively, for 18 hours under normoxic (20% oxygen) and hypoxic (1% oxygen)
conditions. The indicated concentrations of ferrous ethylenediammonium sulfate (A)
and AlCl3 (B) were added at the beginning of the experiment. After preparation of cell
extracts and determination of the luciferase activities and protein contents, the results
were expressed as luciferase activities in relative light units per microgram of cellular
protein. Means ⫾ SD of 3 independent experiments.
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BLOOD, 15 AUGUST 2000 䡠 VOLUME 96, NUMBER 4
EPOLONES ACTIVATE HIF-1␣
1563
that 3 mol of CPX are necessary to chelate 1 mol of iron, whereas 1
mol of DFX is sufficient for the same purpose.
Discussion
Figure 8. Iron blocks the DFX- and CPX-mediated HIF-1␣ protein induction in
HepG2 cells. HepG2 cells were treated with CPX (8 ␮mol/L), DFX (100 ␮mol/L), and
2,2⬘dipyridyl (100 ␮mol/L) for 4 hours in the presence or absence of a 1:1 molar ratio
of simultaneously added ferrous ethylenediammonium sulfate. HIF-1␣ protein was
quantitated by immunoblotting as described in Figure 4.
concentration in the cell culture medium. Using a colorimetric
assay with a lower iron detection limit of 0.4 ␮mol/L (see
“Materials and methods”), we determined an iron concentration in
the FCS of 148 ⫾ 19 ␮mol/L, whereas the iron concentration in the
DMEM itself was 1.4 ⫾ 0.35 ␮mol/L and, hence, close to the
detection limit. Therefore, the medium containing 10% FCS
contains 16-␮mol/L iron. In conclusion, iron in the cell culture
medium can be completely chelated with DFX but not CPX at their
respective optimal concentrations, especially considering the fact
Figure 9. Spectrophotometric analysis of the iron chelation stoichiometry of DFX
and CPX. (A) Spectra of DFX and CPX solutions in water (100 ␮mol/L) with or without
100-␮mol/L ferrous ethylenediammonium sulfate. (B) Relationship between iron:
chelator molar ratio and relative absorption at 430:226 nm19 and 421:247 nm for DFX
and CPX, respectively. The chelator concentrations were held constant at 100 ␮mol/L
in the presence of the indicated molar ratios of ferrous ethylenediammonium sulfate.
In this study, we describe the construction of the HRCHO5
hypoxia-reporter cell line, which contains a stably integrated
hypoxia-responsive luciferase reporter gene under the control of 6
HBSs. This allows easy and rapid monitoring of the activity of
HIF-1. However, it cannot be formally excluded that one of the
other HIF ␣ family members, HIF-2␣20-23 or HIF-3␣,24 might also
be involved in the activation of reporter gene expression, because
they display features very similar to HIF-1␣.25,26 Originally thought
to be expressed specifically in endothelial cells, HIF-2␣ has
recently been found in many other cell lines of nonendothelial
origin.25 Thus, CHO cells might also express HIF-2␣ or even
HIF-3␣, the expression pattern of the latter not being known yet.
We therefore specified our results by detecting HIF-1␣ in a human
cell line (HepG2) using the monoclonal antihuman HIF-1␣ antibody mgc3 that does not cross-react with the other family
members.14
The functioning of the HRCHO5 cell line has been validated by
stimulation with hypoxia as well as the known hypoxia-mimetics
Co2⫹, Ni2⫹, and DFX.5,11,27 Interestingly, Co2⫹ and DFX, but not
Ni2⫹, showed additive effects on HRCHO5 reporter gene activity
when combined with hypoxia. We previously reported that hypoxia
and Co2⫹ also had additive effects on Epo secretion in hepatoma
cell lines,28 which is not in agreement with other reports.5 Our
results imply that Co2⫹ and DFX do not only interfere with the
putative oxygen sensor but might have additional positive effects
on the oxygen signaling pathway, for example, associated with
reactive oxygen species production by a localized Fenton reaction
probably involved in oxygen sensing and signaling.29,30
Using the HRCHO5 cell line, we identified the 2 fungal
epolones,2,3 pycnidione and 8-methyl-pyridoxatin, as well as the
related pyridone, CPX, as potent inducers of HIF-1␣ activation.
Most of the studies in this work were performed with the epolone
CPX, which has been chosen because of its relatively simple
molecular structure and because it is commercially available at a
low price. Clinically, CPX is used as an antimycoticum in
dermatologic and vaginal creams.31 The mechanisms of its antimicrobial action have not been completely resolved, but CPX seems
to interfere with membrane integrity, a probable HIF-1␣–
independent effect. Apart from the epolones, several other substances were tested in the HRCHO5 cell line, which we suspected
to influence HIF-1 activity: insulin and insulin-like growth factors I
and II32-34; interleukin-1␣, interleukin-1␤, tumor necrosis factor␣35; lipopolysaccharide36; tumor growth factor-␤37,38; ferrous ethylenediammonium sulfate, FeCl2, FeCl311,39; ZnCl240; the angiogenesis inhibitor epigallocatechingallate found in drinking tea41; and
taurin.42 These agents have been reported either to be induced by
hypoxia or to interfere with the expression of hypoxia-inducible
genes. However, none of these agents affected HIF-1–dependent
reporter gene activity in normoxic or hypoxic HRCHO5 cells (data
not shown).
The epolones attenuated luciferase activity in HRCHO5 cells
when added at concentrations above 16 ␮mol/L under normoxic
conditions and above 4 ␮mol/L under hypoxic conditions. We
attribute this effect to a putative cytotoxicity of the epolones, which
might increase when combined with the additional stress of
exposure to hypoxia. Consistent with this notion, we observed a
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1564
BLOOD, 15 AUGUST 2000 䡠 VOLUME 96, NUMBER 4
WANNER et al
detachment of the cells with a concomitant decrease of cellular
reporter gene activity after treatment with higher doses of epolones
(data not shown). In support of this idea, it has been reported that
CPX (as well as DFX) can block the cell cycle at the G1/S phase
boundary.43,44 HIF-1␣ protein induction in HepG2 cells by hypoxia
and epolones (Figure 4) was diminished compared with the
corresponding luciferase activities in HRCHO5 cells (Figure 2).
The reason for this finding is not completely clear but might be
related to the presence of other members of the HIF family that
contribute to reporter gene activation or be related to a higher
sensitivity of HepG2 cells to hypoxic cell culture conditions than
CHO cells.
Interestingly, the function of both the established iron chelator
DFX, a sideramine obtained from Streptomyces pilosus,17 and of
CPX could be blocked dosage dependently by adding ferrous iron
salts. Ferrous iron is rapidly oxidized by ambient oxygen yielding
the DFX-chelatable ferric iron. We hence do not know whether
ferrous or ferric iron preferentially inhibits CPX-mediated HIF-1␣
activation. Because we discovered that CPX is an iron-dependent
chromophore, we could directly determine the iron chelation
stoichiometry to be 1:3 iron:CPX. This is in agreement with the
presence of 1 hydroxamic acid group in CPX (bidentate) compared
with 3 such groups in DFX (hexadentate), the latter chelating iron
at a 1:1 stoichiometry.
The well-characterized, HIF-1␣–inducing iron chelators DFX11
and 2,2⬘-dipyridyl18 are thought to mimic hypoxia by displacing
iron from the porphyrin ring of the putative heme oxygen sensor5,39
or by removing iron from the reactive oxygen species-generating
Fenton reaction.29,30 Therefore, CPX could induce HIF-1–
dependent gene expression via similar mechanisms. The finding
that AlCl3 also blocked CPX-induced reporter gene activity suggests that epolones (as well as DFX) potentially might induce
HIF-1␣ by chelation of a metal other than iron. This would have
important implications for the nature of the oxygen sensor, but
further experiments will be required to establish such a mechanism.
While 50- to 100-␮mol/L DFX is necessary to maximally
induce HIF-1–dependent reporter gene expression, the epolones
already show maximal activation at 4 to 8 ␮mol/L. Because we
found an iron concentration in the cell culture medium of 16
␮mol/L, the concentration of DFX, but not of CPX, would be
sufficient to completely chelate total iron in the medium. Considering that a 3-fold higher molarity of the bidentate CPX than of the
hexadentate DFX is required to chelate the same quantity of iron,
and regarding the fact that 8-methyl-pyridoxatin is even 10 times
more potent than CPX (5-fold induction of Epo gene expression at
0.3 ␮mol/L compared with 3-␮mol/L CPX),2 we conclude that the
epolones cannot activate HIF-1␣ simply by chelating all iron in the
medium as might be the case for DFX.
The hexadentate iron chelator DFX has been reported to be
ineffective as an intracellular iron chelator (at concentrations
similar to those used in our study), whereas the lipophilic bidentate
hydroxypyridinone class of iron chelators efficiently chelated
intracellular iron.45 In analogy, differences between the cellular
permeability of DFX and epolones could explain their different
concentration optima. Indeed, the bidentate iron chelator 1,2-diethyl3-hydroxypyridin-4-1 (CP-94) induced VEGF messenger RNA
(mRNA) expression in Hep3B hepatoma cells already at 10
␮mol/L.37 However, CP-94 did not induce Epo mRNA at 10
␮mol/L, and the highest VEGF and Epo mRNA induction was
found at 200 ␮mol/L, which is clearly different from our results
with the epolones.
In conclusion, fundamental differences appear to exist between
CPX and other known hypoxia-mimicking iron chelators with
respect to their structure; metal ion preference; iron chelation
stoichiometry, affinity, and kinetics; cellular uptake; intracellular
stability; and ability to chelate the intracellular labile iron pool.
Therefore, differences might also exist in their interaction with the
putative oxygen sensor iron center as well as their interference with
the oxygen signaling pathway. Better understanding of these differences probably will help in the elucidation of the different steps
involved in the regulation of oxygen-dependent gene expression.
Acknowledgments
We are grateful to Peter J. Nielsen for the gift of CHO cells,
Franziska Parpan for technical assistance, and Christian Gasser for
the artwork.
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2000 96: 1558-1565
Epolones induce erythropoietin expression via hypoxia-inducible
factor-1 α activation
Roger M. Wanner, Patrick Spielmann, Deborah M. Stroka, Gieri Camenisch, Isabelle Camenisch,
Annette Scheid, David R. Houck, Christian Bauer, Max Gassmann and Roland H. Wenger
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