Portal de Periódicos da CAPES

Oxygen-regulated Transferrin Expression Is Mediated by Hypoxia-inducible Factor-1

1997; Elsevier BV; Volume: 272; Issue: 32 Linguagem: Inglês

10.1074/jbc.272.32.20055

1083-351X

Andreas Rolfs, Ivica Kvietikova, Max Gassmann, Roland H. Wenger,

Metabolism, Diabetes, and Cancer

Transferrin (Tf) is a liver-derived iron transport protein whose plasma concentration increases following exposure to hypoxia. Here, we present a cell culture model capable of expressing Tf mRNA in an oxygen-dependent manner. A 4-kilobase pair Tf promoter/enhancer fragment as well as the 300-base pair liver-specific Tf enhancer alone conveyed hypoxia responsiveness to a heterologous reporter gene construct in hepatoma but not HeLa cells. Within this enhancer, a 32-base pair hypoxia-responsive element was identified, which contained two hypoxia-inducible factor-1 (HIF-1) binding sites (HBSs). Mutation analysis showed that both HBSs function as oxygen-regulated enhancers in Tf-expressing as well as in non-Tf-expressing cell lines. Mutation of both HBSs was necessary to completely abolish hypoxic reporter gene activation. Transient co-expression of the two HIF-1 subunits HIF-1α and aryl hydrocarbon receptor nuclear translocator (ARNT)/HIF-1β resulted in enhanced reporter gene expression even under normoxic conditions. Overexpression of a dominant-negative ARNT/HIF-1β mutant reduced hypoxic activation. DNA binding studies using nuclear extracts from the mouse hepatoma cell line Hepa1 and the ARNT/HIF-1β-deficient subline Hepa1C4, as well as antibodies raised against HIF-1α and ARNT/HIF-1β confirmed that HIF-1 binds the Tf HBSs. Mutation analysis and competition experiments suggested that the 5′ HBS was more efficient in binding HIF-1 than the 3′ HBS. Finally, hypoxic induction of endogenous Tf mRNA was abrogated in Hepa1C4 cells, confirming that HIF-1 confers oxygen regulation of Tf gene expression by binding to the two HBSs present in the Tf enhancer. Transferrin (Tf) is a liver-derived iron transport protein whose plasma concentration increases following exposure to hypoxia. Here, we present a cell culture model capable of expressing Tf mRNA in an oxygen-dependent manner. A 4-kilobase pair Tf promoter/enhancer fragment as well as the 300-base pair liver-specific Tf enhancer alone conveyed hypoxia responsiveness to a heterologous reporter gene construct in hepatoma but not HeLa cells. Within this enhancer, a 32-base pair hypoxia-responsive element was identified, which contained two hypoxia-inducible factor-1 (HIF-1) binding sites (HBSs). Mutation analysis showed that both HBSs function as oxygen-regulated enhancers in Tf-expressing as well as in non-Tf-expressing cell lines. Mutation of both HBSs was necessary to completely abolish hypoxic reporter gene activation. Transient co-expression of the two HIF-1 subunits HIF-1α and aryl hydrocarbon receptor nuclear translocator (ARNT)/HIF-1β resulted in enhanced reporter gene expression even under normoxic conditions. Overexpression of a dominant-negative ARNT/HIF-1β mutant reduced hypoxic activation. DNA binding studies using nuclear extracts from the mouse hepatoma cell line Hepa1 and the ARNT/HIF-1β-deficient subline Hepa1C4, as well as antibodies raised against HIF-1α and ARNT/HIF-1β confirmed that HIF-1 binds the Tf HBSs. Mutation analysis and competition experiments suggested that the 5′ HBS was more efficient in binding HIF-1 than the 3′ HBS. Finally, hypoxic induction of endogenous Tf mRNA was abrogated in Hepa1C4 cells, confirming that HIF-1 confers oxygen regulation of Tf gene expression by binding to the two HBSs present in the Tf enhancer. Iron is an essential trace metal in all living organisms. Both iron overload and iron depletion can severely affect physiological processes such as development, erythropoiesis, or biochemical metabolism (reviewed in Refs. 1Bonkovsky H.L. Am. J. Med. Sci. 1991; 301: 32-43Crossref PubMed Scopus (141) Google Scholar and 2Finch C. Blood. 1994; 84: 1697-1702Crossref PubMed Google Scholar). The liver represents the major organ of iron storage in the body and is most susceptible to injuries due to iron overload (1Bonkovsky H.L. Am. J. Med. Sci. 1991; 301: 32-43Crossref PubMed Scopus (141) Google Scholar). Thus, iron hemostasis has to be tightly balanced, and, as a consequence, free iron occurs only transiently in the serum. When iron is absorbed from the small intestine into the blood, it immediately binds apotransferrin to form transferrin (Tf) 1The abbreviations used are: Tf, transferrin; AhR, aryl hydrocarbon receptor; ARNT, AhR nuclear translocator; EMSA, electrophoretic mobility shift assay; Epo, erythropoietin; HBS, HIF-1 binding site; HIF, hypoxia-inducible factor; VEGF, vascular endothelial growth factor; bp, base pair(s); kb, kilobase pair(s). 1The abbreviations used are: Tf, transferrin; AhR, aryl hydrocarbon receptor; ARNT, AhR nuclear translocator; EMSA, electrophoretic mobility shift assay; Epo, erythropoietin; HBS, HIF-1 binding site; HIF, hypoxia-inducible factor; VEGF, vascular endothelial growth factor; bp, base pair(s); kb, kilobase pair(s)., which is then transported by the plasma to all tissues of the vertebrate's body. Delivery of iron occurs by binding of Tf to the Tf receptor followed by endocytosis. In erythroblasts, iron is primarily required for heme synthesis in mitochondria. Tissue-specific expression of the Tf gene is controlled by distinct positive and negative regulatory elements located 5′ to the transcription initiation site. Apart from the promoter, the best studied element within this region is the −3600/−3300 enhancer (hereafter referred to as the Tf enhancer). Thiscis-acting element enhances the activity of the Tf promoter in human Hep3B hepatoma cells in a tissue-specific manner (3Boissier F. Augé-Gouillou C. Schaeffer E. Zakin M.M. J. Biol. Chem. 1991; 266: 9822-9828Abstract Full Text PDF PubMed Google Scholar, 4Sawaya B.E. Aunis D. Schaeffer E. J. Neurosci. Res. 1996; 43: 261-272Crossref PubMed Scopus (16) Google Scholar). Studies in Hep3B and HeLa cells revealed that multiple liver-enriched and ubiquitous factors interact with the Tf enhancer (3Boissier F. Augé-Gouillou C. Schaeffer E. Zakin M.M. J. Biol. Chem. 1991; 266: 9822-9828Abstract Full Text PDF PubMed Google Scholar, 5Augé-Gouillou C. Petropoulos I. Zakin M.M. FEBS Lett. 1993; 323: 4-10Crossref PubMed Scopus (28) Google Scholar, 6Petropoulos I. Augé-Gouillou C. Zakin M.M. J. Biol. Chem. 1991; 266: 24220-24225Abstract Full Text PDF PubMed Google Scholar). The Tf enhancer, however, is inactive in Tf-expressing neuronal and Sertoli cells (4Sawaya B.E. Aunis D. Schaeffer E. J. Neurosci. Res. 1996; 43: 261-272Crossref PubMed Scopus (16) Google Scholar, 5Augé-Gouillou C. Petropoulos I. Zakin M.M. FEBS Lett. 1993; 323: 4-10Crossref PubMed Scopus (28) Google Scholar). Hypoxia, a reduction in oxygen concentration, is increasingly recognized as an important regulator of gene expression (reviewed in Ref. 7Bunn H.F. Poyton R.O. Physiol. Rev. 1996; 76: 839-885Crossref PubMed Scopus (1037) Google Scholar). The best established example of oxygen-regulated gene expression is provided by the erythropoietic growth factor erythropoietin (Epo, reviewed in Ref. 8Jelkmann W. Physiol. Rev. 1992; 72: 449-489Crossref PubMed Scopus (998) Google Scholar). The two human hepatoma cell lines HepG2 and Hep3B are so far the only permanent cell culture models available to investigate oxygen-regulated Epo expression (9Goldberg M.A. Glass G.A. Cunningham J.M. Bunn H.F. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 7972-7976Crossref PubMed Scopus (296) Google Scholar). Apart from Epo, we recently demonstrated hypoxic induction of several acute phase genes in HepG2 cells (10Wenger R.H. Rolfs A. Marti H.H. Bauer C. Gassmann M. J. Biol. Chem. 1995; 270: 27865-27870Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). Acute phase reactants are liver-derived serum proteins whose production is induced by proinflammatory cytokines (reviewed in Ref. 11Heinrich P.C. Castell J.V. Andus T. Biochem. J. 1990; 165: 621-636Crossref Scopus (2262) Google Scholar). Tf expression was of particular interest since this protein is one of the rare examples of acute phase reactants that are down-regulated during the acute phase response in both human serum and HepG2 cells (11Heinrich P.C. Castell J.V. Andus T. Biochem. J. 1990; 165: 621-636Crossref Scopus (2262) Google Scholar). In contrast, we found a marked increase in Tf transcription following hypoxic (1% O2) culture of HepG2 cells (10Wenger R.H. Rolfs A. Marti H.H. Bauer C. Gassmann M. J. Biol. Chem. 1995; 270: 27865-27870Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar), suggesting that different signaling pathways are mediating the effects of these two stimuli. Given that the erythroid marrow uses more than 80% of plasma iron (2Finch C. Blood. 1994; 84: 1697-1702Crossref PubMed Google Scholar), and considering that hypoxia increases erythropoiesis, it is conceivable that an increase in plasma iron transport capacity is required for hypoxia-induced Epo-mediated erythropoiesis. Indeed, hypoxia was shown to increase iron absorption (12Mendel G.A. Blood. 1961; 18: 727-736Crossref PubMed Google Scholar), and hypoxic up-regulation of Tf serum protein concentrations has previously been found in mice (13Simpson R.J. Ann. Hematol. 1992; 65: 260-264Crossref PubMed Scopus (23) Google Scholar) and rats (14Colehour J.K. Borsook H. Graybiel A. Am. J. Physiol. 1957; 191: 113-114Crossref PubMed Scopus (4) Google Scholar, 15Osterloh K.R.S. Simpson R.J. Snape S. Peters T.J. Blut. 1987; 55: 421-431Crossref PubMed Scopus (18) Google Scholar) exposed to hypobaric hypoxia (0.5 atm) for 1–3 days. Although some of these experiments were established some 40 years ago, the molecular mechanisms leading to hypoxically enhanced Tf expression have not been unraveled so far, mainly due to the lack of a suitable cell culture model. The hypoxia-inducible factor-1 (HIF-1) was originally identified by its ability to bind to a hypoxia-responsive cis-element located 3′ to the Epo gene (16Semenza G.L. Wang G.L. Mol. Cell. Biol. 1992; 12: 5447-5454Crossref PubMed Scopus (2137) Google Scholar). HIF-1 is a heterodimer consisting of an α and a β subunit, both belonging to the basic-helix-loop-helix-Per-AhR/ARNT-Sim family of transcription factors (17Wang G.L. Jiang B.-H. Rue E.A. Semenza G.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5510-5514Crossref PubMed Scopus (4928) Google Scholar). Whereas the α subunit is a novel member of this family, the β subunit is identical to the aryl hydrocarbon receptor nuclear translocator (ARNT) known to heterodimerize with the aryl hydrocarbon receptor/dioxin receptor (AhR) following ligand binding (reviewed in Refs. 7Bunn H.F. Poyton R.O. Physiol. Rev. 1996; 76: 839-885Crossref PubMed Scopus (1037) Google Scholar and 18Wenger R.H. Gassmann M. Biol. Chem. 1997; 378: 609-616PubMed Google Scholar). We have established cell culture models to study oxygen-dependent Tf expression and have subsequently analyzed the regulation of the Tf enhancer. Our results demonstrate the presence of two HIF-1 binding sites (HBSs) within the Tf enhancer and show that binding of HIF-1 to these sites confers oxygen-regulated Tf gene expression. The human hepatoma cell lines Hep3B and HepG2 were obtained from American Type Culture Collection (ATCC numbers HB-8064 and HB-8065, respectively). The mouse hepatoma cell lines Hepa1 (also termed Hepa1c1c7) and Hepa1C4 (19Gradin K. McGuire J. Wenger R.H. Kvietikova I. Whitelaw M.L. Toftgård R. Tora L. Gassmann M. Poellinger L. Mol. Cell. Biol. 1996; 16: 5221-5231Crossref PubMed Scopus (380) Google Scholar) were kind gifts of L. Poellinger (Karolinska Institute, Stockholm, Sweden). All cells were cultured in Dulbecco's modified Eagle's medium (high glucose, Life Technologies, Inc.) supplemented with 10% heat-inactivated fetal calf serum (Boehringer Mannheim), 100 units/ml penicillin, 100 μg/ml streptomycin, 1 × non-essential amino acids, 2 mml-glutamine, and 1 mmsodium pyruvate (all Life Technologies, Inc.) in a humidified atmosphere containing 5% CO2 at 37 °C. Oxygen tensions in the incubator (Forma Scientific, model 3319) were either 140 mm Hg (20% O2, v/v, normoxia) or 7 mm Hg (1% O2, v/v, hypoxia). Cells were subjected to hypoxic induction at a cell density of 2 × 105 cells/cm2. The human epitheloid carcinoma cell line HeLaS3 (ATCC CCL-2.2) was cultured in suspension in Ham's F-12 medium (Life Technologies, Inc.) supplemented as described above. Hypoxic induction was achieved as described elsewhere (20Jiang B.-H. Semenza G.L. Bauer C. Marti H.H. Am. J. Physiol. 1996; 271: C1172-C1180Crossref PubMed Google Scholar). Briefly, HeLaS3 cells were incubated at a density of 1 × 107 cells/ml in an IL 237 tonometer (Instrumentation Laboratory) under continuous stirring for 4 h at 37 °C using gas mixtures of either 20% O2, 5% CO2, and 75% N2 (normoxia), or 1% O2, 5% CO2 and 94% N2 (hypoxia) at a flow rate of 500 ml/min. Immediately following stimulation, RNA was isolated as described by Chomczynski and Sacchi (21Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162 (2nd Ed.): 156-159Crossref PubMed Scopus (62909) Google Scholar). Total RNA (10 μg) was denatured in formamide/formaldehyde and electrophoresed through a 1% agarose gel containing 6% formaldehyde as described (22Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Following pressure blotting (Stratagene) to nylon membranes (Biodyne A, Pall) and UV cross-linking (Stratalinker, Stratagene), the filters were hybridized to cDNA probes labeled with [α-32P]dCTP to a specific activity of 1 × 109 dpm/μg using the random-primed DNA labeling method (22Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Hybridization was performed in 50% formamide, 10% dextran sulfate, 5 × Denhardt's solution, 200 μg/ml sonicated salmon sperm DNA, 1% SDS, 0.9 m NaCl, 60 mmNaH2PO4, 6 mm EDTA (pH 7.0) for 14 h at 42 °C. The filters were washed to a final stringency of 55 °C in 0.1 × SSC, 0.2% SDS and the signals recorded using a PhosphorImager (Molecular Dynamics). The Tf, α1-antitrypsin, β-actin, ribosomal protein L28, and 28 S ribosomal RNA cDNA probes were obtained as described previously (10Wenger R.H. Rolfs A. Marti H.H. Bauer C. Gassmann M. J. Biol. Chem. 1995; 270: 27865-27870Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 23Wenger R.H. Marti H.H. Schuerer-Maly C.C. Kvietikova I. Bauer C. Gassmann M. Maly F.E. Blood. 1996; 87: 756-761Crossref PubMed Google Scholar). All probes were purified free of vector sequences by restriction digestion and agarose gel purification. pGLTf4000 was constructed by insertion of the 4-kb KpnI-BamHI fragment from the Tf promoter/enhancer-containing plasmid pTfCAT (kindly provided by M. M. Zakin, Institut Pasteur, Paris, France) into theKpnI-BglII sites of pGL3Basic (Promega). Luciferase reporter gene constructs containing the heterologous SV40 promoter were obtained by inserting the DNA sequences of interest into the BamHI site 3′ to the luciferase gene of the pGL3Promoter plasmid (Promega). The liver-specific Tf enhancer from nucleotide position −3.6 kb to −3.3 kb relative to the transcriptional start site (3Boissier F. Augé-Gouillou C. Schaeffer E. Zakin M.M. J. Biol. Chem. 1991; 266: 9822-9828Abstract Full Text PDF PubMed Google Scholar) was obtained by polymerase chain reaction amplification of genomic DNA using the oligonucleotide primers 5′-GGTCAGGCAGAGGACACTG-3′ and 5′-CAGTTCTAGACCAACCCAAG-3′. The oligonucleotides containing wild type and mutated HBSs are shown in Fig. 4. Copy number and orientation were determined using RVprimer4 (Promega) by T7 polymerase-mediated single-stranded DNA sequencing following the manufacturer's instructions (Pharmacia Biotech Inc.). The β-galactosidase expression vector pCMVlacZ was a kind gift of S. Kozlov (Institute of Biochemistry, Zürich, Switzerland). The HIF-1α (pCMVhHIF-1α) and ARNT/HIF-1β (pCMVhARNT and pCMVΔbARNT) expression vectors (19Gradin K. McGuire J. Wenger R.H. Kvietikova I. Whitelaw M.L. Toftgård R. Tora L. Gassmann M. Poellinger L. Mol. Cell. Biol. 1996; 16: 5221-5231Crossref PubMed Scopus (380) Google Scholar) were generously provided by L. Poellinger. Hep3B, HepG2, and HeLa cells (0.2–1 × 107 in 350 μl of medium without fetal calf serum) were co-transfected with 25 μg each of luciferase and β-galactosidase reporter gene constructs by electroporation at 250 V and 960 microfarads (Gene Pulser, Bio-Rad). After recovering, the cells were split in two aliquots and incubated for 36 h at 20% or 1% O2, respectively. After washing twice with phosphate-buffered saline, the cells were lysed in reporter lysis buffer (Promega) and luciferase and β-galactosidase activities were determined according to the manufacturer's instructions (Promega) using a Biocounter M1500 luminometer (Lumac) and a DU-62 spectrophotometer (Beckman), respectively. Differences in the transfection efficiency and extract preparation were corrected by normalization to the corresponding β-galactosidase activities. Luciferase activities were expressed relative to the empty parental vector (pGL3Basic or pGL3Promoter) transfectants. For transient overexpression assays in Hep3B cells, 10 μg of each expression vector was co-transfected together with equal amounts of the luciferase reporter construct pTfHBSww and the control plasmid pCMVlacZ. The unrelated vector plasmid pBluescript (Stratagene) was added to adjust the total amount of DNA per electroporation to 50 μg. Nuclear extracts were prepared as described previously (24Kvietikova I. Wenger R.H. Marti H.H. Gassmann M. Nucleic Acids Res. 1995; 23: 4542-4550Crossref PubMed Scopus (191) Google Scholar). Briefly, 1 × 108 cells were washed twice with ice-cold phosphate-buffered saline and once with buffer A (10 mm Tris-HCl (pH 7.8), 1.5 mmMgCl2, 10 mm KCl). After incubation on ice for 10 min, the cells were lysed by 10 strokes of a Dounce homogenizer, and the nuclei were pelleted and resuspended in buffer C (420 mm KCl, 20 mm Tris-HCl (pH 7.8), 1.5 mm MgCl2, 20% glycerol) and incubated at 4 °C for 30 min with gentle agitation. Immediately before use, buffers A and C were supplemented with 0.5 mmdithiothreitol, 0.4 mm phenylmethylsulfonyl fluoride, 2 μg/ml each of leupeptin, pepstatin, and aprotinin, and 1 mm Na3VO4 (all obtained from Sigma). The nuclear extract was centrifuged, and the supernatant was dialyzed twice against buffer D (20 mm Tris-HCl (pH 7.8), 100 mm KCl, 0.2 mm EDTA, 20% glycerol). Protein concentrations were determined using the Bradford protein assay (Bio-Rad) with bovine serum albumin as standard. Sequences of the oligonucleotide probes used for EMSA are shown in Fig. 4. The EPOHBS oligonucleotides have been described previously (24Kvietikova I. Wenger R.H. Marti H.H. Gassmann M. Nucleic Acids Res. 1995; 23: 4542-4550Crossref PubMed Scopus (191) Google Scholar). All oligonucleotides (Microsynth) were purified on 10% polyacrylamide gels prior to 5′ end-labeling of the sense strand with [γ-32P]ATP (Hartmann) using T4-polynucleotide kinase (Fermentas). Unincorporated nucleotides were removed by gel filtration over Bio-Gel P60 (fine) columns (Bio-Rad). Labeled sense strands were annealed to a 2-fold molar excess of unlabeled antisense strands. DNA-protein binding reactions were carried out for 20 min at 4 °C in a total volume of 20 μl containing 4–5 μg of nuclear extract, 0.1–0.4 μg of sonicated, denatured calf thymus DNA (Sigma), and 1 × 104 cpm of oligonucleotide probe in 10 mm Tris-HCl (pH 7.5), 50 mm KCl, 50 mm NaCl, 1 mm MgCl2, 1 mm EDTA, 5 mm dithiothreitol, and 5% glycerol and run on 4% non-denaturing polyacrylamide gels. Electrophoresis was performed at 200 V in TBE buffer (89 mm Tris, 89 mm boric acid, 5 mm EDTA) at 4 °C, and dried gels were autoradiographed. For supershift analysis, each 1 μl of rabbit polyclonal antisera derived against HIF-1α or ARNT/HIF-1β (kind gift of L. Poellinger) was added to the completed EMSA reaction mixture and incubated for 16 h at 4 °C prior to loading. For competition experiments, a 4–500-fold molar excess of unlabeled annealed oligonucleotides was added to the binding reaction prior to addition of labeled probes. We previously reported on oxygen-regulated mRNA expression of several acute phase genes in the human hepatoma cell line HepG2 (10Wenger R.H. Rolfs A. Marti H.H. Bauer C. Gassmann M. J. Biol. Chem. 1995; 270: 27865-27870Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). Regulation of the Tf gene was of special interest since Tf transcription was down-regulated in response to proinflammatory cytokines (e.g. interleukin-6), but was up-regulated following exposure to low oxygen concentrations. To test whether hypoxic Tf induction observed in HepG2 cells might represent a general phenomenon in liver cells, we also exposed Hep3B cells, another human hepatoma cell line, to 1% O2 for 1–3 days. As shown by RNA blotting experiments, Tf mRNA was up-regulated about 1.5-fold in Hep3B cells (Fig. 1 A) and up to 4.5-fold in HepG2 cells (Fig. 1 B). A similar hypoxic induction pattern of endogenous gene expression in the two cell lines was observed for the acute phase reactant α1-antitrypsin, which was included as positive control (Fig. 1, A andB). Specificity of hypoxic up-regulation was shown using L28 and 28 S control hybridizations since β-actin mRNA was also slightly up-regulated in both hepatoma cell lines and thus not suitable as a normalization probe (10Wenger R.H. Rolfs A. Marti H.H. Bauer C. Gassmann M. J. Biol. Chem. 1995; 270: 27865-27870Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar).Figure 2Hypoxia responsiveness of the Tf 5′ flanking region. A, structural organization of the 5′ flanking region of the Tf gene (according to Ref. 4Sawaya B.E. Aunis D. Schaeffer E. J. Neurosci. Res. 1996; 43: 261-272Crossref PubMed Scopus (16) Google Scholar). B, luciferase reporter gene activity following transient transfection and hypoxic induction for 36 h of Hep3B, HepG2, and HeLa cells with luciferase expression plasmids containing the 4-kb Tf promoter/enhancer. A co-transfected β-galactosidase expression vector served as internal control for transfection efficiency and extract preparation. All values were normalized to the normoxic luciferase activities obtained with the empty pGL3Basic vector which were arbitrarily defined as 1. Means ± S.D. of three to four independent experiments are shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 1RNA blot analysis of hypoxic Tf mRNA induction. Human Hep3B (A) and HepG2 (B) hepatoma cell lines were exposed to hypoxia as indicated. Hybridization with cDNA probes derived from the ribosomal protein L28 or the ribosomal RNA 28 S served as controls for equal loading and blotting efficiency.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In a first attempt to identify Tf regulatory sequences conveying hypoxia-inducible Tf transcription, a −4000 to +39 (numbering according to Ref. 3Boissier F. Augé-Gouillou C. Schaeffer E. Zakin M.M. J. Biol. Chem. 1991; 266: 9822-9828Abstract Full Text PDF PubMed Google Scholar) Tf promoter/enhancer DNA fragment (Fig.2 A) was inserted upstream of a promoterless luciferase reporter gene vector. Following transient transfection into Tf-expressing Hep3B and HepG2 cells, as well as into non-Tf-expressing HeLa cells, this 4-kb Tf promoter/enhancer induced basal luciferase expression 10-, 26-, and 8-fold in Hep3B, HepG2 and HeLa cells, respectively (Fig. 2 B, open bars). Hypoxia (1% O2) stimulated luciferase expression 4.1- and 5.6-fold in Hep3B and HepG2 cells, respectively, but no significant hypoxic induction could be observed in HeLa cells (Fig. 2 B,filled bars). Thus, hypoxia responsiveness seems to be coupled to liver-specific cis-acting elements present within this Tf promoter/enhancer DNA fragment. In analogy to the liver-specific enhancer and the hypoxia-responsive element residing in close vicinity in the Epo 3′ flanking region (16Semenza G.L. Wang G.L. Mol. Cell. Biol. 1992; 12: 5447-5454Crossref PubMed Scopus (2137) Google Scholar), we wondered whether the −3600/−3300 bp liver-specific Tf enhancer (Fig. 2 A) might be responsible for oxygen responsiveness of the Tf gene. To test this, we subcloned the 300-bp Tf enhancer downstream of a luciferase reporter gene driven by a heterologous SV40 promoter. The Tf enhancer induced normoxic luciferase expression by 8.1-, 1.6-, and 2.0-fold in Hep3B, HepG2, and HeLa cells, respectively (Fig. 3, open bars). This expression level was further up-regulated by exposing the cells to hypoxia; luciferase activity in Hep3B, HepG2, and HeLa cells increased 3.1-, 6.8-, and 1.8-fold, respectively (Fig. 3, filled bars). The weak hypoxic inducibility (1.4-fold) of the pGL3Promoter plasmid itself has been reported previously (24Kvietikova I. Wenger R.H. Marti H.H. Gassmann M. Nucleic Acids Res. 1995; 23: 4542-4550Crossref PubMed Scopus (191) Google Scholar). Thus, similar to the observations using the 4-kb Tf promoter/enhancer, the 300-bp Tf enhancer alone conferred hypoxia inducibility in Hep3B and HepG2 hepatoma cells, but was not significantly active in non-Tf-expressing HeLa cells. A computer-assisted search using a HIF-1 consensus DNA-binding site (24Kvietikova I. Wenger R.H. Marti H.H. Gassmann M. Nucleic Acids Res. 1995; 23: 4542-4550Crossref PubMed Scopus (191) Google Scholar) as query revealed the presence of two tandemly arrayed putative HBSs beginning at nucleotide positions 174 and 191, respectively (Fig. 4), within the 300-bp Tf enhancer (numbering according to Ref. 3Boissier F. Augé-Gouillou C. Schaeffer E. Zakin M.M. J. Biol. Chem. 1991; 266: 9822-9828Abstract Full Text PDF PubMed Google Scholar). No other matches to the HIF-1 query were found in the published nucleotide sequences of the Tf gene. To test whether these two putative HBSs were functionally oxygen-responsive, we synthesized oligonucleotides containing both sites in either the wild type configuration (TfHBSww), or with one (TfHBSwm or TfHBSmw) or both (TfHBSmm) HBS sitesmutated (Fig. 4). Single copies of these oligonucleotides were inserted 3′ to a luciferase reporter gene driven by a heterologous SV40 promoter. For comparison, a hypoxia-responsive luciferase construct (pGLEPOHBS.3) containing three concatamerized copies of the Epo HBS was included in this study (24Kvietikova I. Wenger R.H. Marti H.H. Gassmann M. Nucleic Acids Res. 1995; 23: 4542-4550Crossref PubMed Scopus (191) Google Scholar). Luciferase activity was determined following transient transfection of Hep3B and HeLa cells, splitting in two aliquots and 36 h of normoxic or hypoxic cell culture. Compared with the normoxic control cells, hypoxia increased luciferase expression from the control plasmid (pGLEPOHBS.3) 4.1- and 6.8-fold in Hep3B and HeLa cells, respectively (Fig.5). Hypoxic induction mediated by the two tandemly arrayed, putative Tf HBSs (pGLTfHBSww) was more effective in Hep3B cells (9.4-fold) than in HeLa cells (3.7-fold). Although again less pronounced in HeLa cells compared with Hep3B cells (similar to the 300-bp Tf enhancer; see Fig. 3), the putative Tf HBSs functioned as hypoxia-dependent enhancer in both cell lines that do or do not express Tf (Fig. 5). This observation is reminiscent of the Epo HBS, which has previously been reported to enhance hypoxic gene expression in Epo-expressing and non-Epo-expressing cell lines (25Maxwell P.H. Pugh C.W. Ratcliffe P.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2423-2427Crossref PubMed Scopus (357) Google Scholar,26Wang G.L. Semenza G.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4304-4308Crossref PubMed Scopus (1176) Google Scholar). Mutation of either one of the two putative Tf HBSs (plasmids pGLTfHBSwm and pGLTfHBSmw) only partially reduced hypoxic luciferase expression, and a double mutation of both sites (plasmid pGLTfHBSmm) was necessary to completely abrogate oxygen responsiveness down to the basal level observed with the empty vector alone (Fig. 5). To investigate the involvement of the HIF-1 protein complex in Tf regulation, we performed transient expression experiments using the HIF-1α and/or ARNT/HIF-1β expression vectors pCMVhHIF-1α and pCMVhARNT, respectively (19Gradin K. McGuire J. Wenger R.H. Kvietikova I. Whitelaw M.L. Toftgård R. Tora L. Gassmann M. Poellinger L. Mol. Cell. Biol. 1996; 16: 5221-5231Crossref PubMed Scopus (380) Google Scholar). They were co-transfected into Hep3B cells together with the reporter gene construct pGLTfHBSww (depicted in Fig.5), the normalization plasmid pCMVlacZ and the unrelated plasmid pBluescript used to equalize the total amount of DNA per transfection. As shown in Fig. 6(open bars), transient overexpression, under normoxic conditions, of either of the two HIF-1 subunits weakly (about 2-fold) induced reporter gene expression, whereas expression of both HIF-1 subunits induced luciferase expression by 5.8-fold (Fig. 6, open bars). Co-expression with a reporter gene construct containing mutant HBSs (pGLTfHBSmm) did not result in enhanced luciferase expression (data not shown), implying that HIF-1 needs to bind to the Tf HBSs to transactivate reporter gene expression. Hypoxia also activated the Tf HBSs (Fig. 6, filled bars), and overexpression of the two HIF-1 subunits further enhanced this effect 1.8-fold. Interestingly, overexpression of a dominant negative ARNT/HIF-1β mutant (pCMVΔbARNT), which lacks the basic domain and hence is still capable of heterodimerizing with HIF-1α but cannot bind DNA (19Gradin K. McGuire J. Wenger R.H. Kv