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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Wanner, R. M. Articles by Wenger, R. H. Search for Related Content PubMed PubMed Citation Articles by Wanner, R. M. Articles by Wenger, R. H. Related Collections Red Cells Gene Expression Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, 15 August 2000, Vol. 96, No. 4, pp. 1558-1565 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 From the Institute of Physiology, University of Zürich-Irchel, Zürich, Switzerland; and OSI Pharmaceuticals, MYCOsearch, Durham, NC.     Abstract Top Abstract Introduction Materials and methods Results Discussion References Induction of erythropoietin (Epo) expression under hypoxic conditions is mediated by the heterodimeric hypoxia-inducible factor (HIF)-1. Following binding to the 3' hypoxia-response element (HRE) of the Epo gene, HIF-1 markedly enhances Epo transcription. To facilitate the search for HIF-1 (ant)agonists, a hypoxia-reporter 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 µmol/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 µmol/L) than CPX were required 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 µmol/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 Top Abstract Introduction Materials and methods Results Discussion References 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 hypoxia-inducible 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 hypoxia-dependent 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 hypoxia-reporter cell line, investigated the effects of epolones on HIF-1 activation, and unraveled the mechanism by which epolones activate Epo expression under normoxic conditions.     Materials and methods Top Abstract Introduction Materials and methods Results Discussion References 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-methyl-pyridoxatin) 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-µg/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-µL medium without FCS were mixed with 50 µg of DNA in 50 µL of 10-mmol/L Tris-HCl (pH 7.4), and 1-mmol/L ethylenediaminetetraacetic acid and electroporated at 250 V and 960 µF (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 µg) of nuclear extracts were fractionated by 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrotransferred 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 0.625-mmol/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-µL samples by treatment with 50 µL of 142-mmol/L KMnO4 and 600-mmol/L HCl at 60°C for 2 hours. Thereafter, 10 µL 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-µg samples of iron-saturated purified transferrin (Life Technologies).     Results Top Abstract Introduction Materials and methods Results Discussion References 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+ dose-dependently induced luciferase activity under normoxic conditions, approximately reaching the hypoxic values at concentrations of 50 µmol/L (CoCl2) and 100 µmol/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 µmol/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). View larger version (22K): [in this window] [in a new window]   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. 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, 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 µmol/L (16 µmol/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. View larger version (24K): [in this window] [in a new window]   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 epolonesCPX, pycnidione, and 8-methyl-pyridoxatinfor 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. 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 conferred neither CPX nor hypoxic induction of reporter gene expression, suggesting that both stimuli activate reporter gene expression via similar mechanisms. View larger version (20K): [in this window] [in a new window]   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 µmol/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. 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 normoxic 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. View larger version (59K): [in this window] [in a new window]   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 µmol/L) as well as with CoCl2 (100 µmol/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 View larger version (127K): [in this window] [in a new window]   Figure 5. Immunofluorescence analysis of HIF-1 expression in HepG2 cells. HepG2 cells were treated with hypoxia (1% oxygen) and/or CPX (8 µmol/L) for 4 hours, and HIF-1 was detected using the monoclonal antibody mgc314 followed by a fluorescein isothiocyanate-conjugated secondary antibody. View larger version (79K): [in this window] [in a new window]   Figure 6. EMSA of HepG2 nuclear extracts. HepG2 cells were treated for 4 hours with DFX (100 µmol/L) or CPX (8 µmol/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 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. View larger version (18K): [in this window] [in a new window]   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 µmol/L) and CPX (8 µmol/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. 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 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 µmol/L, whereas 50-µmol/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 View larger version (61K): [in this window] [in a new window]   Figure 8. Iron blocks the DFX- and CPX-mediated HIF-1 protein induction in HepG2 cells. HepG2 cells were treated with CPX (8 µmol/L), DFX (100 µmol/L), and 2,2'dipyridyl (100 µmol/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. 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 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. View larger version (21K): [in this window] [in a new window]   Figure 9. Spectrophotometric analysis of the iron chelation stoichiometry of DFX and CPX. (A) Spectra of DFX and CPX solutions in water (100 µmol/L) with or without 100-µmol/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 µmol/L in the presence of the indicated molar ratios of ferrous ethylenediammonium sulfate. In this context, it would be important to know the iron concentration in the cell culture medium. Using a colorimetric assay with a lower iron detection limit of 0.4 µmol/L (see "Materials and methods"), we determined an iron concentration in the FCS of 148 ± 19 µmol/L, whereas the iron concentration in the DMEM itself was 1.4 ± 0.35 µmol/L and, hence, close to the detection limit. Therefore, the medium containing 10% FCS contains 16-µmol/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 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 Top Abstract Introduction Materials and methods Results Discussion References 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-220-23 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 µmol/L under normoxic conditions and above 4 µmol/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 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-µmol/L DFX is necessary to maximally induce HIF-1-dependent reporter gene expression, the epolones already show maximal activation at 4 to 8 µmol/L. Because we found an iron concentration in the cell culture medium of 16 µmol/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 µmol/L compared with 3-µmol/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-diethyl-3-hydroxypyridin-4-1 (CP-94) induced VEGF messenger RNA (mRNA) expression in Hep3B hepatoma cells already at 10 µmol/L.37 However, CP-94 did not induce Epo mRNA at 10 µmol/L, and the highest VEGF and Epo mRNA induction was found at 200 µmol/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.     Footnotes 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. 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: Roland H. Wenger, Institut für Physiologie, Medizinische Universität zu Lübeck, Ratzeburger Allee 160, D-23538 Lübeck, Germany; e-mail: wenger{at}physio.mu-luebeck.de.     References Top Abstract Introduction Materials and methods Results Discussion References 1. Jelkmann W. Erythropoietin: structure, control of production, and function. Physiol Rev. 1992;72:449-489[Free Full Text]. 2. 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