|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
Blood, 15 June 2005, Vol. 105, No. 12, pp. 4613-4619. Prepublished online as a Blood First Edition Paper on March 1, 2005; DOI 10.1182/blood-2004-10-3980.
HEMATOPOIESIS Copper-dependent activation of hypoxia-inducible factor (HIF)-1: implications for ceruloplasmin regulationFrom the Carl-Ludwig-Institute of Physiology, University of Leipzig, Germany; Institute of Physiology, University of Lübeck, Germany; Cell Physiology Group, Medical Faculty, Martin-Luther-University Halle, Germany; Institute for Cardiovascular Research, Bayer HealthCare AG, Wuppertal, Germany; 5th Lerner Research Institute, Cleveland Clinic Foundation, OH; and the Institute of Physiology, University of Zürich, Switzerland.
Cellular oxygen partial pressure is sensed by a family of prolyl-4-hydroxylase domain (PHD) enzymes that modify hypoxia-inducible factor (HIF)  subunits. Upon hydroxylation under normoxic conditions, HIF is bound by the von Hippel-Lindau tumor suppressor protein and targeted for proteasomal destruction. Since PHD activity is dependent on oxygen and ferrous iron, HIF-1 mediates not only oxygen- but also iron-regulated transcriptional gene expression. Here we show that copper (CuCl2) stabilizes nuclear HIF-1 under normoxic conditions, resulting in hypoxia-response element (HRE)-dependent reporter gene expression. In in vitro hydroxylation assays CuCl2 inhibited prolyl-4-hydroxylation independently of the iron concentration. Ceruloplasmin, the main copper transport protein in the plasma and a known HIF-1 target in vitro, was also induced in vivo in the liver of hypoxic mice. Both hypoxia and CuCl2 increased ceruloplasmin (as well as vascular endothelial growth factor [VEGF] and glucose transporter 1 [Glut-1]) mRNA levels in hepatoma cells, which was due to transcriptional induction of the ceruloplasmin gene (CP) promoter. In conclusion, our data suggest that PHD/HIF/HRE-dependent gene regulation can serve as a sensory system not only for oxygen and iron but also for copper metabolism, regulating the oxygen-, iron- and copper-binding transport proteins hemoglobin, transferrin, and ceruloplasmin, respectively. (Blood. 2005;105:4613-4619)
The hypoxia-inducible factor 1 (HIF-1) is an ubiquitously expressed transcriptional master regulator of many genes regulating mammalian oxygen homeostasis.1 Among others, the corresponding gene products are involved in erythropoiesis, iron metabolism, angiogenesis, control of blood flow, glucose uptake and glycolysis, pH regulation, and cell-cycle control.2 HIF-1 is a 1 1 heterodimer specifically recognizing the HIF-binding site within cis-regulatory hypoxia response elements.3 Under normoxic conditions, the von Hippel-Lindau tumor suppressor protein (pVHL) targets the HIF-1 subunit for rapid ubiquitination and proteasomal degradation.4 Binding of the pVHL tumor suppressor protein requires the modification of HIF-1 by prolyl-4-hydroxylation at prolines 402 and 564 of human HIF-1.5-8 A family of 3 oxygen- and iron-dependent prolyl-4-hydroxylases called PHD1, PHD2, PHD3, or HPH3, HPH2, HPH1, respectively, has been shown to hydroxylate HIF.9,10 A fourth member, called PH-4, regulates HIF-1 in overexpression conditions only.11 Thus, limited oxygen supply prevents HIF hydroxylation and degradation.12 This unusual mechanism of protein regulation provides the basis for the very rapid HIF-1 response to hypoxia.13 In addition to protein stability, oxygen-dependent C-terminal asparagine hydroxylation of HIF-1 by factor inhibiting HIF (FIH) prevents transcriptional cofactor recruitment, thereby fine-tuning HIF-1 activity following a further decrease in oxygen availability.14,15 Among the HIF-1 targets are the genes encoding transferrin, transferrin receptor, heme oxygenase-1, and ceruloplasmin, which coordinately regulate iron metabolism.16-20 Increased iron uptake, release from the liver, plasma transport, and uptake in the bone marrow are essential to sustain the erythropoietic function of erythropoietin, the prototype HIF-1 target. Ceruloplasmin is a multicopper plasma protein containing ferroxidase activity necessary for Fe3+ saturation of transferrin.21 Hereditary aceruloplasminemia in humans as well as targeted deletion of the ceruloplasmin gene (Cp) in mice results in iron metabolism disorders characterized by anemia, hepatic iron overload, and neurodegeneration, demonstrating a tight connection between copper and iron metabolism.22-26
Iron deficiency has been known for more than a decade to induce erythropoietin gene expression and HIF-1
Cell lines and cell culture All cell lines were cultured in Dulbecco modified Eagle medium (high glucose) as described previously.29 Oxygen partial pressures in the hypoxic workstation (InVivO2-400; Ruskinn Technology, Leeds, United Kingdom) or in the incubator (Model 3319; Forma Scientific, Illkirch, France) were either 140 mmHg (20% O2 vol/vol, normoxia) or 7 mmHg (1% O2 vol/vol, hypoxia). Stable transfection with the plasmid pH3SVL led to HIF-dependent luciferase reporter cell lines designated HRCHO5 (derived from CHO Chinese hamster ovary cells) or HRB5 (derived from Hep3B human hepatoma cells), respectively, as described before.30-32 The luciferase reporter gene pH3SVL is driven by a simian virus 40 (SV40) promoter and contains a total of 6 HIF-1 DNA-binding sites derived from the transferrin gene.16 Cell proliferation/viability assays Cell proliferation/viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) conversion assay as described before.33 Absorbances at 570 nm were determined in a 96-well photometer and noncellular background was subtracted. Data were normalized to the MTT conversion activity of solvent-treated normoxic control cells, which were arbitrarily defined as 1. Reporter gene assays Stably transfected hypoxia reporter cell lines were seeded in triplicates into 96-well dishes at a density of 104/well. After incubation overnight, freshly dissolved CuCl2 or CuCl in phosphate-buffered saline (PBS) was added at the concentrations indicated and the cells were exposed to normoxic or hypoxic conditions for 24 hours. In some experiments, the kinase inhibitors LY294002, PD98059, or PD169316 (Calbiochem, VWR International, Schwalbach, Germany) dissolved in dimethyl sulfoxide (DMSO), or DMSO alone, were added in addition to 100 µM CuCl2. Firefly luciferase reporter gene activity was determined as described by the manufacturer (Promega, Madison, WI) using a microplate luminometer (Berthold, Regensdorf, Switzerland). Results were normalized to the solvent-treated normoxic control values, which were arbitrarily defined as 1. Transient transfections Hep3B cells were transiently transfected with the CP promoter firefly reporter construct pGL-Cp477420 by the calcium phosphate coprecipitation method.34 CP enhancer constructs, either containing a wild-type or a mutant HRE, were cloned into pGL3Prom (Promega) as described previously.20 Semiconfluent cells on a 10-cm dish were cotransfected with 20 µg of pGL-Cp4774 together with 0.2 µg of the renilla luciferase control construct pRL-SV40 (Promega) for 16 hours. Cells were detached with 2 mM EDTA (ethylenediaminetetraacetic acid; pH 8.0) in PBS, distributed onto 2 24-well dishes, and allowed to recover for 8 hours. Freshly prepared CuCl2 in PBS or PBS alone was added and the cells were incubated for 24 hours under normoxic or hypoxic conditions. Luciferase activities were determined using the dual-luciferase kit (Promega) as described previously. Protein extraction and immunoblot analyses
Cells were treated with CuCl2 or the iron chelator ciclopirox olamine (CPX)32 at the concentrations indicated for 6 or 24 hours. For stability experiments, 20 µg/mL cycloheximide was added following 6 hours hypoxic induction with or without CuCl2 and the cells were allowed to reoxygenate. Combined cytoplasmic and nuclear extracts of cultured cells were prepared using 0.4 M NaCl and 0.1% Nonidet P-40 (NP-40) in extraction buffer as described previously.29 Protein concentrations were determined by the Bradford method using bovine serum albumin (BSA) as a standard.35 For immunoblot analysis, cellular protein (100 µg or 50 µg in stability experiments) was electrophoresed through 5% sodium dodecyl sulfate (SDS)-polyacrylamide gels and electrotransferred onto nitrocellulose membranes (Amersham, Freiburg, Germany) by semidry blotting (BioRad, München, Germany). Membranes were stained with Ponceau S (Sigma, Buchs, Switzerland) to confirm equal protein loading and transfer. HIF-1
HIF-1
Immunofluorescence analysis was performed as described before.29 Briefly, cells were fixed with formaldehyde for 10 minutes, washed with PBS, permeabilized with Triton X-100, and rinsed again with PBS. After blocking nonspecific binding sites with 3% BSA in PBS for 30 minutes, the cells were incubated for 1 hour with mouse monoclonal anti-HIF-1 In vitro prolyl-4-hydroxylation assay
Prolyl-4-hydroxylation activity was determined as recently described in detail elsewhere.36 Briefly, a biotinylated peptide containing Pro564 of HIF-1 mRNA quantification Exposure of mice in triplicate to 7.5% oxygen for 0, 24, 48, and 72 hours and excision of their livers was described earlier.38 Total RNA from frozen mouse liver tissue or cultured Hep3B cells was isolated and analyzed by Northern blotting as described previously.16 Hybridization probes were obtained by restriction digestion and gel isolation followed by labeling with the random-primed method.16 The mouse ceruloplasmin cDNA39 was a kind gift of J. Gitlin (St Louis, MO) and the ribosomal protein L28 cDNA was cloned from a HepG2 cDNA library.40 Radioactive signals were detected by phosphoimaging and quantitated using QuantityOne software (Bio-Rad). Human ceruloplasmin, vascular endothelial growth factor (VEGF), glucose transporter-1 (Glut-1) and L28 were determined by real-time polymerase chain reaction (PCR). Briefly, 2 µg of total RNA was reverse transcribed with Superscript III according to the manufacturer's instructions (Invitrogen, Basel, Switzerland). Real-time PCR was performed in duplicates with 1% of the cDNA reaction mixture using a SybrGreen Q-PCR reagent kit on a MX3000P PCR cycler according to the manufacturer's instructions (Stratagene, Amsterdam, the Netherlands). Dilution series of the corresponding plasmids were used to obtain standard curves. Primers: ceruloplasmin, hCpfwd 5'-gattaattggccccctgatt-3' and hCprev 5'-tgcattgtgaggccttgtag-3'; VEGF, hVEGF165fwd 5'-gaggagggcagaatcatcac-3' and hVEGF165rev 5'-aggcccacagggattttcttgtc-3'; Glut-1, hGlut1fwd 5'-tcactgtgctcctggttctg-3' and hGlut1rev 5'-cctgtgctcctgagagatcc-3'; and L28, hL285.1 5'-gcatctgcaatggatggt-3' and hL283.1 5'-tgttcttgcggatcatgtgt-3'.
CuCl2 can activate a hypoxia-response element-containing reporter gene We previously established a Chinese hamster ovary (CHO) cell line, called HRCHO5, which was stably transfected with a HIF-1-dependent luciferase reporter gene containing multiple hypoxia-response elements (HREs).30 Apart from hypoxia, reporter activity in these cells could also be induced by cobalt and nickel salts as well as by iron chelation.30,32 Unexpectedly, we found that CuCl2 dose-dependently induced reporter gene induction approximately 4.1-fold at 500 µM CuCl2 in CHO cells (Figure 1A). The human hepatoma Hep3B cell line is known to synthesize the plasma proteins transferrin and ceruloplasmin, both of which are induced in a HIF-dependent manner upon iron depletion of these cells.16,20 We thus stably transfected Hep3B cells with the HIF-dependent reporter gene like previously used to generate the HRCHO5 cell line. When the resulting cell line (termed HRB5) was treated with various doses of CuCl2 for 24 hours, reporter gene expression increased up to 14-fold at 125 µM CuCl2 (Figure 1B, top graph). Hypoxia alone (24 hours of 1% O2) induced reporter gene activity 11-fold (Figure 1B, bottom graph) and the combined treatment with 1% O2 and 125 µM CuCl2 resulted in a 118-fold reporter gene induction. The CuCl2 dose-response relationship revealed a relatively narrow window of active CuCl2 concentrations. Since we suspected CuCl2 cytotoxicity at higher concentrations, MTT conversion assays were performed to analyze cell viability (ie, the combined decrease in cell growth and/or the increase in cell death). A dose-dependent reduction of MTT conversion activity was found in both cell lines (Figure 1A-B). At CuCl2 concentrations that maximally induced reporter gene expression, MTT conversion was still 60% in CHO and 67% in Hep3B cells, respectively, when compared with untreated cells. Hypoxic conditions did not significantly alter MTT conversion activity in Hep3B cells. In addition to Cu2+, Cu+ also was able to induce reporter gene activity (Figure 1C), which might be due to the disproportionation of cuprous ions to cupric ions and copper in aqueous solutions. Other metal ions tested (Al3+, Ca2+, Fe2+, Fe3+, Gd2+, Mg2+, Mn2+, Se2+, and Zn2+) did not alter reporter gene activity in this type of assay (data not shown).
Nuclear HIF-1 protein induction by CuCl2
The most likely explanation for the induction of reporter gene expression by CuCl2 is the activation of the HIF system. We therefore analyzed whether CuCl2 treatment leads to the stabilization of the HIF-1
We next determined whether CuCl2 leads to a nuclear translocation of stabilized HIF-1 protein. Therefore, the Hep3B subline HRB5 was treated with 1% O2 or 125 µM CuCl2 for 24 hours. As shown by indirect immunofluorescence analysis, HIF-1 protein accumulated exclusively within the nucleus, excluding the nucleoli, following hypoxia or CuCl2 treatment but not in normoxic control cells (Figure 2C).
CuCl2 stabilizes HIF-1
To gain more insight into the molecular mechanisms by which CuCl2 induces and activates HIF-1
In contrast to the kinase pathways that induce HIF-1
CuCl2 inhibits HIF-1
CuCl2-dependent stabilization of HIF-1 To gain more insight into the mechanism by which CuCl2 inhibits PHD activity, we repeated the prolyl-4-hydroxylation assays in the presence of limited concentrations (1 µM) or a vast excess (100 µM) of FeSO4. As shown in Figure 4B, PHD3 activity was inhibited by CuCl2 with an IC50 value of approximately 2.3 µM. However, the inhibitory concentration at 50% (IC50) value did not significantly change when Fe2+ was present in excess, suggesting that iron oxidation and/or competitive replacement from the active center is not the cause for CuCl2-dependent inhibition of PHD activity. Induction of ceruloplasmin mRNA by hypoxia and Cu2+
Cu2+-dependent induction of HIF-1
We next determined mRNA concentrations in cultured Hep3B cells (subline HRB5) exposed to hypoxic conditions for up to 3 days by real-time reverse transcription (RT)-PCR. As shown in Figure 6, ceruloplasmin as well as the prototype oxygen-regulated VEGF and Glut-1, but not L28 control mRNA, are induced by hypoxia at all time points examined, confirming that ceruloplasmin is hypoxia-inducible in Hep3B cells. Of note, 150 µM CuCl2 also induced ceruloplasmin, VEGF, and Glut-1, but not L28 mRNA, in Hep3B cells and the combination with hypoxia further enhanced these mRNA levels (Figure 6). Maximal ceruloplasmin induction by CuCl2 was 5.5-fold after 72 hours of treatment under normoxic conditions.
The ceruloplasmin gene promoter is activated by Cu2+
Cu2+-dependent HIF-1 The involvement of HIF-1 was further analyzed by using the HRE-containing fragment of the ceruloplasmin -3429 to -3639 upstream region linked to the constitutive SV40 promoter. As shown in Figure 7B, the presence of this fragment was sufficient to induce firefly luciferase activity approximately 5-fold by 150 µM CuCl2 or 1% O2 after 24 hours of treatment. Point mutation of this HRE reduced but did not completely inhibit induction by CuCl2 or hypoxia in these experiments, suggesting that other elements might also be involved in CuCl2-dependent activation of ceruloplasmin gene expression.
Molecular mimicry of hypoxia by distinct transition metal ions has been known since 1988, when Bunn and coworkers reported that HepG2 and Hep3B hepatoma cell lines increase erythropoietin gene expression not only under hypoxic conditions but also following treatment with Co2+ or Ni2+ and to a lesser extent Mn2+ salts.41 However, Cu2+ has not been found to induce erythropoietin gene expression in these early experiments. In our study, we demonstrated that Cu2+ can functionally stabilize HIF-1 , leading to target gene induction in hepatoma cells by a mechanism likely to involve the inhibition of prolyl-4-hydroxylation.
Apart from Co2+, Ni2+, or Cu2+, several other transition metals have been reported to induce HIF-1 Spectroscopic and structural studies on 2-oxoglutarate-dependent dioxygenases showed that Cu2+ can substitute active-site Fe2+ in this class of enzymes.12 However, under our experimental conditions Cu2+ does probably not compete for the active site of HIF-PHD3 because even the presence of a vast excess of ferrous iron did not reverse the inhibitory function of CuCl2. Although it cannot be excluded that Cu2+ ions bind to the active site of PHD with considerably higher affinity compared with Fe2+, it appears to be more likely that other, yet unknown mechanisms are responsible for the inhibition of HIF PHDs by transition metal ions.
Recently, van Heerden and coworkers demonstrated that rainbow trout exposed to CuSO4 induce HIF-1 Liver-derived ceruloplasmin is the main copper-binding plasma transport protein and functions as a ferroxidase essential for the oxidation of Fe2+ before it can be bound to transferrin as Fe3+.21 Previous studies demonstrated that ceruloplasmin is transcriptionally regulated by HIF-1 under hypoxic conditions in vitro.20 Here, we demonstrated that ceruloplasmin mRNA expression is also induced in the liver of hypoxic mice in vivo. Because both ceruloplasmin and apotransferrin facilitate iron release from liver cells and macrophages,53-56 the coordinate induction of their transcription under hypoxic conditions is likely required to match the increased iron demand of erythropoietic precursor cells in the bone marrow following stimulation by erythropoietin. The finding that Cu2+ inhibits PHD activity provides the intriguing possibility that the concentration of free Cu2+ regulates the expression of its binding protein in the liver in a HIF-dependent manner. Indeed, when CuSO4 was administered intravenously to copper-deficient pigs, an increase in plasma ceruloplasmin was observed which preceded the increase in plasma iron.57 Other known HIF-1 target genes induced by Cu2+ in vivo include VEGF which is increased at wound sites topically treated with CuSO4.58 These data are consistent with our findings and support a physiological role of HIF-1 in Cu2+-dependent gene regulation, extending the sensory functions of the PHD family beyond oxygen sensing.
The authors wish to thank J. D. Gitlin and P. L. Fox for the generous gifts of plasmids, and B. Stier, U. Lang, C. Franke, B. Dübel, K. Krauss, and P. Spielmann for excellent technical assistance.
Submitted October 14, 2004; accepted February 18, 2005.
Prepublished online as Blood First Edition Paper, March 1, 2005; DOI 10.1182/blood-2004-10-3980.
Supported by grants from the Medical Faculty of the University of Leipzig (formel.1-22 to F.M.), Bundesministerium für Bildung und Forschung (BMBF; NBL3-01ZZ0106 to F.M. and NBL3 to D.M.K.), Deutsche Forschungsgemeinschaft (DFG; Ka1269/5-1 to D.M.K.), Schweizerischer Nationalfonds (SNF; 3100A0-104219 to R.H.W.) and Stiftung für wissenschaftliche Forschung an der Universität Zürich (to R.H.W.).
F.M. and T.L. contributed equally to this work.
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, Institute of Physiology, University of Zürich-Irchel, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland; e-mail: roland.wenger{at}access.unizh.ch.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Top
Abstract
Introduction
, and adenosine monophosphate (AMP) kinases have been reported to be involved in transition metal-dependent HIF-1