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Originally published In Press as doi:10.1074/jbc.M000636200 on April 20, 2000

J. Biol. Chem., Vol. 275, Issue 28, 21048-21054, July 14, 2000
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Role of Hypoxia-inducible Factor-1 in Transcriptional Activation of Ceruloplasmin by Iron Deficiency*

Chinmay K. Mukhopadhyay, Barsanjit Mazumder, and Paul L. FoxDagger

From the Department of Cell Biology, The Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195

Received for publication, January 27, 2000, and in revised form, April 10, 2000

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

A role of the copper protein ceruloplasmin (Cp) in iron metabolism is suggested by its ferroxidase activity and by the tissue iron overload in hereditary Cp deficiency patients. In addition, plasma Cp increases markedly in several conditions of anemia, e.g. iron deficiency, hemorrhage, renal failure, sickle cell disease, pregnancy, and inflammation. However, little is known about the cellular and molecular mechanism(s) involved. We have reported that iron chelators increase Cp mRNA expression and protein synthesis in human hepatocarcinoma HepG2 cells. Furthermore, we have shown that the increase in Cp mRNA is due to increased rate of transcription. We here report the results of new studies designed to elucidate the molecular mechanism underlying transcriptional activation of Cp by iron deficiency. The 5'-flanking region of the Cp gene was cloned from a human genomic library. A 4774-base pair segment of the Cp promoter/enhancer driving a luciferase reporter was transfected into HepG2 or Hep3B cells. Iron deficiency or hypoxia increased luciferase activity by 5-10-fold compared with untreated cells. Examination of the sequence showed three pairs of consensus hypoxia-responsive elements (HREs). Deletion and mutation analysis showed that a single HRE was necessary and sufficient for gene activation. The involvement of hypoxia-inducible factor-1 (HIF-1) was shown by gel-shift and supershift experiments that showed HIF-1alpha and HIF-1beta binding to a radiolabeled oligonucleotide containing the Cp promoter HRE. Furthermore, iron deficiency (and hypoxia) did not activate Cp gene expression in Hepa c4 hepatoma cells deficient in HIF-1beta , as shown functionally by the inactivity of a transfected Cp promoter-luciferase construct and by the failure of HIF-1 to bind the Cp HRE in nuclear extracts from these cells. These results are consistent with in vivo findings that iron deficiency increases plasma Cp and provides a molecular mechanism that may help to understand these observations.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Ceruloplasmin (Cp)1 is a 132-kDa copper protein that is abundant in serum (1). Several lines of evidence suggest that Cp has an important role in iron metabolism. First, Cp catalyzes the conversion of Fe2+ to Fe3+ and is the major ferroxidase in plasma. This function is thought to be critical for loading of iron into apo-transferrin (2). Second, Cp is likely to be important in iron transport and homeostasis. A role of Cp in iron release from cells and tissues has been suggested by early organ culture studies (3, 4) and by recent findings of iron deposits in hereditary Cp deficiency patients (5) and in mice with targeted disruption of the Cp gene (6). In contrast, studies in yeast have revealed that a Cp homologue, fet3p, is required for iron uptake into cells (7, 8), and we have shown that Cp stimulates iron uptake by iron-deficient cells of hepatic and erythroid origin (9, 10). A major function of Cp may be to facilitate the net flux of iron between cells and tissues.

Iron status influences the expression of multiple iron-related genes (11). Thus, the final evidence that Cp is involved in iron metabolism comes from observations that alterations in serum iron status are often accompanied by changes in serum Cp (and copper). This observation was first made by Warburg and Krebs (12), who showed that bleeding birds to make them iron-deficient resulted in a 3-6-fold increase in protein-bound copper in plasma. Similar observations have been made in humans: elevated levels of serum copper are observed in anemia due to massive hemorrhage (13), dietary iron deficiency (14), infection or inflammation (15), and pregnancy (16). After the discovery that Cp is the primary copper protein in blood (17), the increase in serum copper was shown to be due to elevated serum Cp in dietary iron deficiency anemia (18, 19), hemorrhagic anemia (20), sickle cell disease (21), renal failure (22), anemia of inflammation (23-25), and anemia associated with pregnancy (26). Whereas the Cp increase in anemia due to pregnancy and inflammation may be influenced by hormone and cytokine levels, the observations on dietary anemia and hemorrhage provide evidence for a direct relationship between iron deficiency and plasma Cp levels.

Despite abundant in vivo evidence that serum Cp increases during anemia, little is known about the underlying molecular mechanism(s). The liver is the major source of plasma Cp in adults (27, 28), and we have shown that treatment of cultured human hepatocarcinoma HepG2 or Hep3B cells with iron chelators markedly increases Cp gene expression and protein synthesis (9). We have also shown that the increase in Cp mRNA is not due to increased transcript stability but rather is due to an increase in the rate of transcription as demonstrated by nuclear run-on experiments (9). There are at least two known mechanisms by which cellular iron deficiency increases gene transcription. Iron deficiency in Saccharomyces cerevisiae activates the AFT1 transcription factor, which in turn up-regulates the entire iron regulon, including the Cp homologue and ferroxidase, FET3 (29); however, a vertebrate homologue of AFT1 has not yet been identified. In vertebrate cells, iron deficiency has been shown to activate hypoxia-inducible factor-1 (HIF-1), a heterodimeric transcription factor complex.

HIF-1 consists of the helix-loop-helix/Per-aryl hydrocarbon receptor nuclear translocator (ARNT)-Sim proteins, HIF-1alpha and HIF-1beta (identical to ARNT) (30). HIF-1alpha is the key regulatory component of the complex because it is absent under normal conditions but is up-regulated by the iron chelator desferrioxamine (and by hypoxia and CoCl2). In contrast, HIF-1beta expression is essentially constitutive and is promiscuous with respect to downstream targets, interacting with several helix-loop-helix/PER-ARVT-Sim proteins in addition to HIF-1alpha . Upon activation, the HIF-1alpha /HIF-1beta dimer binds to hypoxia-responsive elements (HREs) in multiple genes, including several with important functions in iron metabolism, e.g. erythropoietin (Epo) (31), heme oxygenase-1 (32), transferrin (33), and transferrin receptor (34, 35). We here report studies to elucidate the molecular mechanism(s) underlying transcriptional activation of Cp by iron deficiency in hepatic cells, and we report an important role for HIF-1.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Lines and Culture Conditions-- Human hepatoma cell lines Hep3B and HepG2 were from American Type Culture Collection and were cultured in Dulbecco's modified Eagle's medium (Sigma). Mouse hepatoma cell lines Hepa-1c1c7 and Hepa c4 were kind gifts of Oliver Hankinson and were cultured in modified Eagle's medium. All media were supplemented with 10% heat-inactivated fetal bovine serum, 100 units/ml penicillin, 100 mg/ml streptomycin, and 2 mM L-glutamine (Life Technologies, Inc.). All experiments were done with cells at 50-60% confluence. For most experiments, cells were maintained in a humidified atmosphere containing 5% CO2 at 37 °C. For experiments involving hypoxia, oxygen tension in the hypoxia chamber (Billups-Rothenberg, San Diego, CA) was set at either 20% O2 (v/v), for normoxic treatment, or 1% O2 (v/v), for hypoxic treatment.

RNA Blot Analysis-- RNA was isolated from HepG2 or Hep3B cells using Trizol reagent (Life Technologies, Inc.) according to the company's protocol. Total RNA (20 µg) was denatured in formamide/formaldehyde and electrophoresed through a 1% agarose gel containing 6% formaldehyde and was blotted on to nylon membranes (Schleicher & Schuell). After cross-linking with ultraviolet light (Stratalinker, Stratagene), the filters were subjected to hybridization with a 646-base pair BstXI and BamHI fragment of a human Cp cDNA probe (bp 984-1629 of the open reading frame) radiolabeled by random priming with [alpha -32P]dCTP.

Cloning of the 5'-Flanking Region of Human Cp-- To obtain the 5'-flanking region of the human Cp gene, a human genomic library in the bacterial artificial chromosome vector pBeloBAC11 (Research Genetics, Huntsville, AL) was screened by PCR using primers corresponding to the extremes of the Cp exon 1 (bp 1-25 and 117-140 of the open reading frame) (36). A pool of putative positive clones was rescreened by PCR with anchor primers in exon 1, and a single positive clone was isolated. DNA sequencing with an exon 1-specific primer 40 bp downstream of the start codon gave a sequence identical to the known Cp exon 1 sequence, demonstrating its authenticity. To isolate the 5'-flanking region of Cp, purified DNA from the positive clone was treated with restriction enzymes and screened by Southern analysis with a 114-bp Cp exon 1 probe. A 5-kilobase fragment released by NcoI was positive by Southern analysis and was subcloned into the NcoI site of the pGEM-5Zf(+) vector (Promega). The authenticity and direction of the product (pGEM-Cp) was verified by sequencing. The 4774-bp fragment was shown to be authentic by the identity of the 3'-end with the known human Cp exon 1 sequence and by homology to the proximal region of the 5'-flanking region of the rat Cp gene (28, 37).

Construction of Vectors Containing Cp Promoter/Enhancer Fragments-- Cp promoter/enhancer constructs, engineered to contain SacI and XhoI restriction sites, were made by PCR amplification using Pfu polymerase (Stratagene), primers containing these restriction site, and pGEM-Cp as template. The constructs were ligated into SacI and XhoI sites of pGL3basic or pGL3prom vectors (Promega). For the long constructs inserted into pGL3basic, the PCR products from two separate amplification reactions were ligated to form a single construct. In brief, a proximal construct was made from -2389 (just 5'-upstream of an EcoRI site) to -1 (the nucleotide upstream of the translation initiation site, which is here defined as +1). Several distal constructs were PCR-amplified from 5'-termini at -4774, -3789, -3639, and -3576 to the 3'-terminus at -2325. The proximal and distal products were ligated at the EcoRI site and then into the 5'-SacI and 3'-XhoI sites upstream of luciferase in pGL3basic. Other constructs were made by PCR amplification of pGEM-Cp between -3639 and -3429 and between -3639 and -3544 and were ligated into the SacI and XhoI sites of the pGL3prom vector upstream of the SV40 promoter and luciferase. Site-directed mutagenesis of the Cp HRE was done by the megaprimer method (38). All deletion and mutation constructs were verified by sequencing.

Transient Transfection of Cells and Reporter Assays-- HepG2, Hep3B, Hepa-1c1c7, and Hepa c4 cells at about 50% confluence in six-well plates were transiently transfected for 16 h with 2 µg of reporter plasmid using Lipofectin (Life Technologies, Inc.) according to the manufacturer's directions. To monitor transfection efficiencies, 0.25 µg of SV40-beta -galactosidase reporter gene was co-transfected. Transfected cells were allowed to recover for 6 h in Dulbecco's modified Eagle's medium with 10% fetal bovine serum, and then cells were incubated under the described experimental conditions for 16 h. Luciferase (Promega) and beta -galactosidase (Tropix, Bedford, MA) activities in cell extracts were determined by chemiluminescence.

Preparation of Nuclear Extracts-- Nuclear extracts were prepared from Hep3B, HepG2, Hepa-1c1c7, and Hepa c4 cells as described (33). Briefly, 1 × 108 cells were washed twice with ice-cold phosphate-buffered saline and once with a solution containing 10 mM Tris-HCl, pH 7.8, 1.5 mM MgCl2, and 10 mM KCl, supplemented with a protease inhibitor mixture containing 0.5 mM dithiothreitol, 0.4 mM phenylmethylsulfonyl fluoride, and 2 µg/ml each of leupeptin, pepstatin, and aprotinin (Sigma). After incubation on ice for 10 min, the cells were lysed by 10 strokes with a Dounce homogenizer, and the nuclei were pelleted and resuspended in a solution containing 420 mM KCl, 20 mM Tris-HCl, pH 7.8, 1.5 mM MgCl2, and 20% glycerol; supplemented with the protease mixture described above; and incubated at 4 °C with gentle agitation. The nuclear extract was centrifuged at 10,000 × g for 10 min, and the supernatant was dialyzed twice against a solution of 20 mM Tris-HCl, pH 7.8, 100 mM KCl, 0.2 mM EDTA, and 20% glycerol. Protein concentration was determined using the Bio-Rad reagent with bovine serum albumin as standard.

Electrophoretic Mobility Shift Assay (EMSA)-- Sequences of the sense strands of the oligonucleotide probes used for EMSA were as follows: 5'-TCT GTA CGT GAC CAC ACT CAC CTC-3' (Cp HRE), 5'-TCT GTA AAA GAC CAC ACT CAC CTC-3' (mutated Cp HRE), and 5'-GCC CTA CGT GCT GTC TCA CAC AGC-3' (Epo HRE). The sense and antisense strands were annealed, gel-purified, and end-labeled with [gamma -32P]ATP (NEN Life Science Products) using T4-polynucleotide kinase (Promega). Unincorporated nucleotide was removed by gel filtration using G-25 Sephadex columns (Quick SpinTM TE, Roche Molecular Biochemicals). To measure DNA-protein interaction, 2-5 × 104 cpm of oligonucleotide probe was incubated with 4-5 µg of nuclear extract and 0.5 µg of sonicated, denatured salmon sperm DNA (Life Technologies, Inc.) in 10 mM Tris-HCl (pH 7.8), 50 mM KCl, 50 mM NaCl, 1 mM MgCl2, 1 mM EDTA, 5 mM dithiothreitol, and 5% glycerol, for 20 min at 4 °C in a total volume of 20 µl. The reaction mixture was subjected to electrophoresis (200 V in 0.3× Tris-buffered EDTA, 4 °C) using 5% nondenaturing polyacrylamide gels. Dried gels were subjected to autoradiography for 16-48 h. For competition experiments, a 10-1000-fold molar excess of unlabeled, annealed oligonucleotide was added to the binding reaction just prior to addition of radiolabeled probe. For gel supershift analysis, 1 µl of rabbit monoclonal antibody against HIF-1alpha or rabbit polyclonal antibody against ARNT/HIF-1beta (both from Novus Biologicals, Littleton, CO) was added after the initial 20-min incubation, and the solution was further incubated for 30 min at 4 °C before electrophoresis.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Regulation of Cp Gene Expression by HIF-1 Agonists-- We have reported that incubation of hepatoma cell lines HepG2 and Hep3B with iron chelators up-regulates Cp mRNA expression by an unidentified transcriptional mechanism (9). To investigate the possible role of HIF-1 in transcriptional regulation of Cp, we tested whether Cp mRNA expression is increased by CoCl2 and hypoxia (1% O2), two agents that transactivate multiple genes via HIF-1 activation (39). Exposure of Hep3B cells to CoCl2 and 1% O2 increased Cp mRNA by about 3.3-fold and 6.0-fold, respectively (Fig. 1); the fold stimulation observed in HepG2 cells was somewhat less: 2.3-fold and 3.5-fold by the same agents. These results are consistent with an important role of HIF-1 in transcriptional activation of Cp.


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  		Fig. 1.   Increase in hepatoma cell Cp mRNA levels by activators of HIF-1. Hep3B (left) and HepG2 (right) cells were treated with medium alone (Cont.), 0.1 mM CoCl2, or 1% O2 (Hpx.) for 16 h. RNA was isolated, and the steady-state level of Cp mRNA was determined by Northern blot analysis of RNA using radiolabeled 646-bp Cp cDNA fragment as a probe (top panels). The 28 S ribosomal RNA subunit was visualized by ultraviolet as a loading and transfer control (bottom panels).

Deletion and Mutation Analysis of Hypoxia-responsive Elements in the Cp Gene 5'-Flanking Region-- To determine whether the Cp gene contains a regulatory sequence(s) responsive to hypoxia and iron deficiency, the 5'-flanking region of the human Cp gene was cloned and tested for activation in a luciferase reporter gene system. Based on previous results that a 732-bp fragment of the rat Cp 5'-flanking region gives maximal transcriptional activity (37), we first cloned and tested a fragment of the human Cp promoter of comparable length (from position -848 to -1). This fragment contained consensus binding sequences for CCAAT/enhancer-binding protein-beta and hepatocyte nuclear factor (HNF)-3beta , transcription factors that are abundant in liver and known to enhance hepatocyte-specific transcription, e.g. cytokine stimulation of acute phase genes (40, 41). The presence of a STAT1 binding site is consistent with the stimulation of Cp gene expression by interferon-gamma in hepatic and monocytic cells (42, 43). The cloned fragment was inserted upstream of luciferase in the promoterless reporter gene vector pGL3basic (Cp-848, -1-Luc) and was transiently transfected into both HepG2 and Hep3B cells. Basal luciferase activity was increased substantially by the promoter fragment (by about 10-15-fold), but exposure of the cells to hypoxia, CoCl2 (not shown), or iron chelators (desferrioxamine or bathophenanthroline sulfate) did not further enhance luciferase expression (Fig. 2A). Longer promoter fragments of 1403, 2389, and 4774 bp were cloned into pGL3basic and similarly tested for transactivation. Cells transfected with constructs containing Cp-1403, -1-Luc or Cp-2389,  -1-Luc did not respond to the HIF-1 agonists (Fig. 2A). Interestingly, these constructs were stimulated by interleukin-6, a classical agonist of the acute phase response, thus showing activity in another context (not shown). The 2389-bp segment contained two consensus binding sites for PU.1, a myeloid-specific transcription factor (44), which may be involved in Cp production by monocyte-macrophages (42, 45). The construct containing the 4774-bp fragment of the 5'-flanking region (Cp-4774, -1-Luc) was stimulated by HIF-1 agonists by at least 5-fold compared with untreated controls in both Hep3B (Fig. 2A) and HepG2 cells (not shown). These results indicate the presence of cis-acting sequences that confer sensitivity to HIF-1 agonists (i.e. HREs) located between positions -4774 and -2390 in the 5'-flanking region of human Cp.


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  		Fig. 2.   Activation of the Cp promoter by iron chelators, CoCl2, and hypoxia: deletion and mutation analysis of Cp gene 5'-flanking region. A, pGL3basic vectors containing the proximal 848, 1403, 2389, or 4774 bp of the Cp gene 5'-flanking region driving luciferase (Luc) were transiently transfected (with a plasmid containing beta -galactosidase to correct for transfection efficiency) into Hep3B cells using Lipofectin. Consensus sequences for transcription factor binding are indicated by arrows (left). After recovery, the cells were treated for 16 h with 1 mM desferrioxamine (DFO), 1 mM bathophenanthroline sulfate (BPS), or 1% O2 or were left untreated (control). Luciferase activity in cell extracts was measured by chemiluminescence and normalized for beta -galactosidase activity (right). B, pGL3basic vectors containing the proximal 4774, 3789, 3639, or 3576 bp of the Cp gene 5'-flanking region driving luciferase were transiently transfected into Hep3B as in A. After recovery, the cells were made iron-deficient by treatment with DFO or BPS, were treated with CoCl2, or were left untreated. Luciferase activity in cell extracts was measured and normalized for beta -galactosidase activity. C, distal segments of the Cp 5'-flanking region were ligated upstream of the SV40 promoter driving luciferase in pGL3prom and were transiently transfected (with a plasmid containing beta -galactosidase) into Hep3B cells as in A. After recovery, the cells were treated for 16 h with DFO, BPS, or 1% O2 or were left untreated. Luciferase activity in cell extracts was measured and normalized by beta -galactosidase activity (right). HIF-1 sites are indicated by open boxes; the HIF-1 mutation site is indicated by × in the box and by an underline below the mutated nucleotides (left).

The presence of consensus HIF-1 binding sites, i.e. (G/A)CGTG (46), in the 5'-flanking region of human Cp was examined. Six consensus sites, arranged in three adjacent pairs, were found between positions -4774 and -2390 (Fig. 2B, left). To determine the activity of these sites, a series of promoter/enhancer fragments was constructed with progressively larger 5'-deletions, and the fragments ligated upstream of luciferase in the promoterless reporter gene pGL3basic. Hep3B cells transfected with a construct in which the upstream pair of consensus HIF-1 binding was removed (Cp-3789, -1-Luc) were still activated by iron chelators or by CoCl2 (Fig. 2B, right). However, transfection of cells with a construct lacking the three upstream-most consensus HIF-1 binding sites (Cp-3576, -1-Luc) resulted in a substantial decrease in basal activity and a complete lack of activation by HIF-1 agonists. This result indicates that the third (from the 5'-end) HIF-1 site is critical for activation. Just upstream of this site is a pair of consensus AP-1 binding sites previously shown to potentiate HIF-1 activation of vascular endothelial growth factor (VEGF) (47). When cells were transfected with a construct lacking the AP-1 sites (Cp-3639, -1-Luc), there was only a small decrease in basal and agonist-stimulated activities, suggesting that these sites do not contribute significantly to Cp gene transactivation. In summary, these results indicated that the 63-bp segment between positions -3639 and -3577 was necessary for Cp transactivation by HIF-1 agonists.

A mutagenesis strategy was used to determine whether the single HRE between -3639 and -3577 was sufficient to confer agonist-stimulated activation. Because adjacent pairs of HREs may be required for maximal transcriptional activation (33, 48), we first examined a region containing the third and fourth (from the 5'-end) consensus HIF-1 binding sites. A 211-bp segment was subcloned upstream of the SV40 promoter driving luciferase in the pGL3prom vector (Cp-3639, -3429-SV40-Luc). Both iron chelators and hypoxia markedly stimulated transactivation of this segment after transfection into Hep3B cells (Fig. 2C). Likewise, the HIF-1 agonists stimulated transcription of a shorter segment containing only the upstream HRE site (Cp-3639, -3544-SV40-Luc), showing that a single HRE element is sufficient for reporter gene transactivation. To demonstrate that the HRE in this enhancer region is responsible for the observed activation, the core 5'-ACGTG-3' sequence of the active HRE in the 211-bp fragment was mutated to 5'-AAAAG-3'. The mutated fragment was not stimulated by either iron chelators or hypoxia. These results suggest that a single HRE present in the Cp enhancer is necessary and sufficient for transactivation by HIF-1 activators, and may be the site of binding of a nuclear transcription factor, most likely HIF-1.

Binding of HIF-1 to the HRE in the Cp Gene 5'-Flanking Region-- EMSAs were used to identify transcription factor complexes that bind to the functional HRE of the Cp enhancer element. Nuclear extracts were prepared from Hep3B or HepG2 cells treated with hypoxia or iron chelators and incubated with a radiolabeled 24-bp probe containing the HRE of human Cp. Specific formation of a radiolabeled complex was observed in cells treated with hypoxia or iron chelators but not in untreated cells (Fig. 3A). Constitutive and nonspecific bands of greater electrophoretic mobility were seen as previously reported for Epo and other HREs (49). As a positive control, extracts from untreated and hypoxic Hep3B cells were incubated with a radiolabeled probe containing the HRE of human Epo; a hypoxia-inducible complex was seen with electrophoretic mobility similar to that observed with the Cp probe. Essentially identical results with Cp were obtained with nuclear extracts from HepG2 cells (not shown). Competition experiments were done to show the specificity of binding of the complex to the Cp HRE probe. The binding of radiolabeled Cp HRE probe to the HIF-1 complex in nuclear extracts from hypoxic Hep3B cells was effectively competed by a 100-fold molar excess of unlabeled Cp HRE probe (Fig. 3B). However, unlabeled competitor probe containing a mutation in the HIF-1 binding site was ineffective even at 1000-fold molar excess. As a positive control, an unlabeled Epo HRE probe was found to be an effective competitor, although perhaps with a lower efficiency than the Cp HRE. Gel supershift studies were done to identify components of the nuclear extract complex that binds to the Cp HRE probe. A rabbit monoclonal antibody against HIF-1alpha shifted the mobility of the complex induced by desferrioxamine DNA-protein binding (Fig. 3C). Furthermore, a polyclonal antibody against ARNT/HIF-1beta completely blocked the formation of the complex. Essentially identical results were observed when the cells were made hypoxic instead of iron-deficient (not shown). Together, these experiments show that HIF-1 agonists induce high affinity binding of HIF-1 to the Cp HRE.


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  		Fig. 3.   Binding of HIF-1 to the HRE of the Cp enhancer (by EMSA). A, Induction of complex formation by HIF-1 agonists. Hep3B cells were exposed for 8 h to 1 mM desferrioxamine (DFO), 1 mM bathophenanthroline sulfate (BPS), or 1% O2 (Hpx.). Nuclear extracts were incubated with 32P-labeled, oligonucleotide 24-mer probes containing either the Cp or Epo HRE. Complexes formed were resolved by 5% nondenaturing polyacrylamide gel electrophoresis and visualized by autoradiography. The positions of the putative HIF-1, constitutive (Const.), and nonspecific (NS) complexes are indicated by arrows. B, competitor binding to show specificity of HIF-1 binding to the Cp enhancer HRE. Hep3B cells were treated with 1% O2 for 9 h, and nuclear extracts were prepared as in A. A 10-, 100-, or 1000-fold molar excess of unlabeled, annealed oligonucleotide competitor representing the wild-type (wt) Cp HRE, the mutant (mut) Cp HRE, or the Epo HRE was added to the nuclear extract reaction mixture just prior to addition of radiolabeled Cp HRE probe. The mutated sequence in the Cp HRE is underlined. C, identification of HIF-1 subunits binding to the Cp enhancer HRE by gel supershift analysis. Hep3B cells were treated with desferrioxamine, and nuclear extracts were prepared as in A. Before subjecting extracts to electrophoresis, the mixtures containing 32P-labeled Cp HRE probe was incubated with 1 µl of anti-HIF-1alpha , anti-HIF-1beta , or both. The supershifted complex is indicated by the open-headed arrow.

HIF-1 Is Required for Transcriptional Activation of Cp by Iron Deficiency and Hypoxia-- To determine whether the HIF-1 complex is required for transcriptional activation of Cp by iron deficiency and hypoxia, we took advantage of the Hepa c4 mouse hepatoma cell line, which is deficient in ARNT/HIF-1beta (50). These cells are derived from the parental Hepa-1c1c7 cell line and have been used by others to show the requirement for HIF-1 in gene transactivation by hypoxia (33). The construct containing the entire cloned Cp promoter enhancer (Cp-4774, -1-Luc) was transiently transfected into both ARNT/HIF-1beta -deficient Hepa c4 cells and the parental Hepa-1c1c7 cells. The luciferase reporter gene was activated by either iron deficiency or hypoxia in the parental cell line but not in the ARNT/HIF-1beta -deficient Hepa c4 cells (Fig. 4A). Essentially identical results were obtained with a construct containing only the active Cp HRE inserted before the SV40 promoter driving luciferase (Cp-3639, -3544-SV40-Luc) (Fig. 4B). To further investigate the specificity of HIF-1 binding to the Cp enhancer, we examined the binding of transcription factor complexes to the Cp HRE in ARNT/HIF-1beta -deficient cells. Binding of the HIF-1 complex to the probe was observed in nuclear extracts made from wild-type Hepa-1c1c7 cells treated with iron chelators or CoCl2; however, essentially no HIF-1 binding was seen in the ARNT/HIF-1beta -deficient cells (Fig. 4C). Together, these results clearly demonstrate the requirement for HIF-1 in activation of Cp transcription by iron chelators and hypoxia.


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  		Fig. 4.   Iron deficiency does not activate Cp promoter in HIF-1beta /ARNT-deficient mouse Hepa c4 cells. Effect of iron chelators on Cp promoter activation in HIF-1beta /ARNT-deficient cells. A, mouse Hepa-1c1c7 (wild-type) and the HIF-1beta /ARNT-deficient variant Hepa c4 cells were transiently transfected with pGL3basic vector containing 4774 bp of the Cp 5'-flanking region upstream of luciferase cDNA (Cp-4774, -1-Luc). The cells were made iron-deficient by incubation with 0.3 mM desferrioxamine (DFO) or 0.3 mM bathophenanthroline sulfate (BPS), were made hypoxic by incubation in 1% O2, or were left untreated (control). Luciferase activity was measured by chemiluminescence in cell extracts, normalized for beta -galactosidase activity, and expressed as fold increase compared with untreated controls. B, both mouse hepatoma lines were transiently transfected with pGL3prom vector containing the Cp HRE upstream of the SV40 promoter driving luciferase cDNA (Cp-3639, -3544-SV40-Luc). Treatment of cells and luciferase activity was measured as in A. C, effect of iron chelators on binding of HIF-1 to the Cp enhancer HRE in HIF-1beta /ARNT-deficient cells. Mouse Hepa-1c1c7 wild-type (left) and Hepa c4 HIF-1beta /ARNT-deficient (right) cells were exposed for 8 h to 0.3 mM DFO, 0.3 mM BPS, or 0.1 mM CoCl2. Nuclear extracts were incubated with 32P-labeled, oligonucleotide 24-mer probes containing the Cp HRE. Complexes formed were resolved by nondenaturing polyacrylamide gel electrophoresis and visualized by autoradiography. The positions of the putative HIF-1 complex, constitutive (Const.), and nonspecific (NS) complexes are indicated by arrows.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The studies here show that Cp is a member of the growing family of genes transcriptionally activated by HIF-1. In support of this conclusion, we found that: (i) multiple HIF-1 agonists, i.e. iron chelators, hypoxia, and CoCl2, all increase Cp mRNA in HepG2 and Hep3B cells, (ii) the 5'-flanking region of the human Cp gene contains an HRE that is necessary and sufficient for agonist-dependent transcriptional activation of a heterologous reporter gene, (iii) a radiolabeled probe containing the human Cp HRE specifically binds to HIF-1 as shown by EMSA and gel supershift studies, and (iv) iron deficiency and hypoxia do not transactivate a Cp promoter-luciferase reporter chimeric gene in mouse hepatoma cells deficient in the ARNT/HIF-1beta subunit of HIF-1. Transcriptional activation of Cp by iron deficiency is functionally significant because Cp protein synthesis is increased by about 3-5-fold in HepG2 cells (9).

The sequence elements required for HIF-1 activation of the Epo gene, the archetype HIF-1-responsive gene, include a core (G/A)CGTG sequence, a downstream CACAG sequence, followed by two consensus HNF-4 binding sites (Fig. 5A). The Cp HRE, like all known HIF-1-responsive elements (46), also contains the critical (G/A)CGTG core sequence (see Fig. 5B for examples). In addition, the Cp HRE contains the consensus CACAG site present in Epo and most, but not all (e.g. human transferrin), HIF-1-responsive enhancers. The Cp HRE also contains a near-consensus (5 out of 6 bp) HNF-4 binding site. This site is not necessary for HIF-1 activation of Epo by hypoxia, but it increases the magnitude of the response. Interestingly, to our knowledge nearby HNF-4 sites have been reported only in the Epo enhancer. The role of the individual cis-acting elements (except for the (G/A)CGTG site) have not yet been defined for Cp; however, the HIF-1-responsive region in the Cp gene appears to have the same tripartite structure as the human Epo gene, and the sequences of the three critical elements are remarkably similar in both genes (14 out of 16 bp). There are important differences between the HREs in Cp and Epo; for example, the Cp HRE is compressed compared with the Epo HRE, and the Epo HRE is located downstream of the open reading frame, whereas the Cp HRE is in the 5'-flanking region.


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  		Fig. 5.   Structure of the Cp hypoxia-responsive element. A, comparison of human Cp and human Epo HRE. HIF-1 binding sites ((G/A)CGTG) are indicated by a double underline, CACAG consensus sequence by a single underline, and HNF-4 sites (TGACCT) by a line above. Conserved nucleotides are shown by vertical lines, and gaps inserted for alignment are shown by dashes. B, the structure of the HRE in multiple HIF-1-responsive genes. The HRE in genes encoding human transferrin receptor (TfR), the complementary (compl.) strand of human transferrin (Tf), human VEGF, human aldolase-A (ALDA), human heme oxygenase-1 (HO-1), and mouse inducible nitric oxide synthase (iNOS) are shown. The consensus HIF-1 binding sites and CACAG sites are indicated as in A.

The genes activated by HIF-1 can be classified into three functional groups. In one group are proteins that increase tissue oxygen delivery by their systemic participation in erythropoiesis, e.g. transferrin (33), transferrin receptor (34, 35), heme oxygenase-1 (32), and erythropoietin (31). The second group contains proteins that increase local oxygen delivery to tissues via modification of blood vessel relaxation and development, e.g. inducible nitric oxide synthesis (51) and VEGF (52, 53). Heme oxygenase-1 produces the potent vasodilator carbon monoxide and may belong to this group in addition to the first. The third group contains proteins that do not alter tissue oxygen delivery but rather are necessary for adaptation of cellular metabolism under conditions of low oxygen, e.g. glucose transporter-1 (54) and most glycolytic enzymes (55). At this time, we can only speculate about the functional group in which Cp belongs. Much experimental evidence suggests that Cp is a member of the first group, i.e. an enhancer of erythropoiesis. First, early studies showed that copper deficiency causes anemia in animals, implicating a copper protein as an important catalyst for hemoglobin formation (56, 57). The role for Cp in erythropoiesis is supported by recent findings in patients with hereditary Cp deficiency who, in general, have microcytic, hypochromic anemia and below-normal hemoglobin level (58-60). The studies with patients have not been confirmed in studies of mice with a disrupted Cp gene in which plasma hemoglobin levels were normal (6). Finally, a role of Cp in erythropoiesis has been shown directly in aplastic anemia patients; injection of purified Cp rapidly and dramatically increases both reticulocyte number and hemoglobin level (61). To our knowledge, Cp is the first positive acute phase reactant protein shown to be regulated by HIF-1 activation. Transferrin is activated by HIF-1 (33) but is a negative acute phase protein. Several (but not all) positive acute phase proteins, e.g. alpha 1-antitrypsin, alpha 1-antichymotrypsin, complement C3, haptoglobin, and alpha 1-acid glycoprotein, are activated by hypoxia, but the specific role of HIF-1 has not been determined (62). Thus, Cp may represent a new functional group of HIF-1 effector proteins, a subset of the proinflammatory acute phase proteins.

Our findings are consistent with many reports that plasma Cp (and copper) is elevated during multiple conditions of anemia including dietary iron deficiency (18, 19), hemorrhage (20), sickle cell disease (21), renal failure (22), pregnancy (26), and anemia of inflammation (23-25). The importance of HIF-1 activation in transcriptional regulation of liver Cp synthesis would suggest that pathological conditions featuring chronic hypoxia will also activate HIF-1 and thus should increase serum Cp. In fact, in patients with various chronic obstructive pulmonary diseases, e.g. chronic bronchitis and asthma, serum Cp is increased by 35-100% compared with controls (63-65). The response is not due to a generalized acute phase response because elevated Cp is not found in a control group of cigarette smokers without pulmonary dysfunction and because two other acute phase reactants, haptoglobin and orosomucoid, are unchanged (63). In studies of healthy young adults, hypoxia induced by exposure to high altitude also significantly increases serum Cp (66).

There remain many unanswered questions about HIF-1 activation of Cp. Despite much effort by several laboratories, the nature of the sensor of iron deficiency and/or hypoxia and the identity of the downstream signal transduction pathway remain elusive. The specific trans-acting factors required for activation of Cp transcription in hepatic cells are yet to be identified. The presence of a near-consensus HNF-4 binding site within the active enhancer of Cp may be significant. In addition, because CBP/p300 is a transcription co-activator that interacts with HIF-1 and HNF-4 to form a complex that binds to the erythropoietin enhancer, the role of CBP/p300 in Cp transactivation should be considered. The in vivo role of HIF-1 in Cp activation certainly must be determined; unfortunately, targeted disruption of the HIF-1 gene results in nonviable mice, and only studies during embryonic development have been reported (67-69). Nonetheless, the results in at least one study have been quite unexpected, namely, that HIF-1-/- mice actually have higher levels of VEGF than control mice, indicating that VEGF levels may be regulated by alternate transcription factor pathways, or possibly by regulation of mRNA stability (70). Given that several genes that control iron homeostasis are regulated by HIF-1, it will be interesting to determine whether other members of this family are similarly regulated, for example, hephaestin, a newly identified, membrane-bound Cp homologue (71). Finally, our understanding of the specific molecular function(s) of Cp in hepatic iron metabolism, erythropoiesis, and the acute phase response (and possibly in cellular copper metabolism and angiogenesis) remains rudimentary. We are hopeful that the present studies on the activation of Cp by HIF-1 will provide clues that will help to elucidate the specific function(s) of Cp during normal and pathophysiological conditions.

    ACKNOWLEDGEMENT

We are grateful to Oliver Hankinson for his generous gift of mouse hepatoma Hepa-1c1c7 and the variant Hepa c4 cells.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant HL29582 (to P. L. F.) and by a Fellowship of the American Heart Association of Northeast Ohio (to C. K. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Cell Biology, The Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195. Tel.: 216-444-8053; Fax: 216-444-9404; E-mail: foxp@ccf.org.

Published, JBC Papers in Press, April 20, 2000, DOI 10.1074/jbc.M000636200

    ABBREVIATIONS

The abbreviations used are: Cp, ceruloplasmin; ARNT, aryl hydrocarbon receptor nuclear translocator; bp, base pair(s); EMSA, electrophoretic mobility shift assay; Epo, erythropoietin; HIF, hypoxia-inducible factor; HNF, hepatocyte nuclear factor; HRE, hypoxia-responsive element; PCR, polymerase chain reaction; VEGF, vascular endothelial growth factor; CREB, cAMP response element-binding protein; CBP, CREB-binding protein.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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