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• W1967855521 abstract "Expression of the transferrin receptor, which mediates iron uptake from transferrin, is negatively regulated post-transcriptionally by intracellular iron through iron-responsive elements in the 3′-untranslated region of the transferrin receptor mRNA. Transcriptional mechanisms are also involved in receptor expression, but these are poorly understood. In this study we have characterized the transferrin receptor promoter region and identified a functional hypoxia response element that contains a binding site for hypoxia-inducible factor-1 (HIF-1). Exposure of K562 and HeLa cells to hypoxia for 16 h resulted in a 2- to 3-fold increase in transferrin receptor mRNA expression. A motif with multipartite organization similar to the hypoxia response element of a number of hypoxia-inducible genes such as erythropoietin was identified within a 100-base pair sequence upstream of the transcriptional start site. Mutation of a site similar to the consensus HIF-binding site (HBS) in this motif attenuated the hypoxic response by 80%. Transient co-expression of the two HIF-1 subunits (HIF-1α and HIF-1β) enhanced the wild type transferrin receptor promoter activity, but that which contained a mutated HBS yielded no such response. Electrophoretic mobility shift assays revealed that HIF-1 was stimulated and bound to the transferrin receptor HBS upon hypoxic challenge. Our results indicate that the transferrin receptor is a target gene for HIF-1. Expression of the transferrin receptor, which mediates iron uptake from transferrin, is negatively regulated post-transcriptionally by intracellular iron through iron-responsive elements in the 3′-untranslated region of the transferrin receptor mRNA. Transcriptional mechanisms are also involved in receptor expression, but these are poorly understood. In this study we have characterized the transferrin receptor promoter region and identified a functional hypoxia response element that contains a binding site for hypoxia-inducible factor-1 (HIF-1). Exposure of K562 and HeLa cells to hypoxia for 16 h resulted in a 2- to 3-fold increase in transferrin receptor mRNA expression. A motif with multipartite organization similar to the hypoxia response element of a number of hypoxia-inducible genes such as erythropoietin was identified within a 100-base pair sequence upstream of the transcriptional start site. Mutation of a site similar to the consensus HIF-binding site (HBS) in this motif attenuated the hypoxic response by 80%. Transient co-expression of the two HIF-1 subunits (HIF-1α and HIF-1β) enhanced the wild type transferrin receptor promoter activity, but that which contained a mutated HBS yielded no such response. Electrophoretic mobility shift assays revealed that HIF-1 was stimulated and bound to the transferrin receptor HBS upon hypoxic challenge. Our results indicate that the transferrin receptor is a target gene for HIF-1. iron-responsive element hypoxia response element hypoxia-inducible factor-1 HIF-binding site iron regulatory protein natural resistance-associated macrophage protein 2 divalent cation transporter-1 erythropoietin vascular endothelial growth factor cyclic AMP-responsive element CRE-binding protein activating transcription factor-1 electrophoretic mobility shift assays reporter plasmids of the transferrin receptor promoter with mutated HBS and CRE, respectively The transferrin receptor is a cell membrane-associated glycoprotein that serves as a gatekeeper in regulating cellular uptake of iron from transferrin, a plasma protein that transports iron in the circulation (1Testa U. Pelosi E. Peschle C. Crit. Rev. Oncog. 1993; 4: 241-276PubMed Google Scholar, 2Ponka P. Lok C.N. Int. J. Biochem. Cell Biol. 1999; (in press)PubMed Google Scholar). Cellular iron uptake from transferrin involves the binding of transferrin to the transferrin receptor, internalization of transferrin within an endocytic vesicle by receptor-mediated endocytosis, and the release of iron from the protein by a decrease in endosomal pH (3Baker E. Morgan E.H. Brock J.H. Halliday J.W. Pippard M.J. Powell L.W. Iron Metabolism in Health and Disease. W. B. Saunders Company, Ltd., London1994: 63-95Google Scholar, 4Richardson D.R. Ponka P. Biochim. Biophys. Acta. 1997; 1331: 1-40Crossref PubMed Scopus (585) Google Scholar). Iron released from transferrin is then transported through the endosomal membrane, and a compelling candidate for serving this function is “natural resistance-associated macrophage protein 2” (Nramp2), also known as DCT1 (divalent cation transporter 1) (5Fleming M.D. Andrews N.C. J. Lab. Clin. Med. 1998; 32: 464-468Abstract Full Text PDF Scopus (36) Google Scholar). After its release from endosomes, iron is transported to intracellular sites of use and/or storage, and the iron-free transferrin that remains receptor-bound returns to the cell surface where apotransferrin is released from the cells (3Baker E. Morgan E.H. Brock J.H. Halliday J.W. Pippard M.J. Powell L.W. Iron Metabolism in Health and Disease. W. B. Saunders Company, Ltd., London1994: 63-95Google Scholar, 4Richardson D.R. Ponka P. Biochim. Biophys. Acta. 1997; 1331: 1-40Crossref PubMed Scopus (585) Google Scholar). With the exception of highly differentiated cells such as erythrocytes, transferrin receptors are probably expressed on all cells, but their levels vary greatly (2Ponka P. Lok C.N. Int. J. Biochem. Cell Biol. 1999; (in press)PubMed Google Scholar, 4Richardson D.R. Ponka P. Biochim. Biophys. Acta. 1997; 1331: 1-40Crossref PubMed Scopus (585) Google Scholar). Transferrin receptors are highly expressed on immature erythroid cells, placental tissue, and rapidly dividing cells, both normal and malignant. In proliferating nonerythroid cells, the expression of transferrin receptors is negatively regulated post-transcriptionally by intracellular iron through iron-responsive elements (IREs)1 in the 3′ untranslated region of the transferrin receptor mRNA. IREs are recognized by specific cytoplasmic proteins (iron regulatory proteins (IRPs)) that, under conditions of decreased iron in the labile pool, bind to the IREs of transferrin receptor mRNA, preventing its degradation. On the other hand, the expansion of the labile iron pool leads to a rapid degradation of transferrin receptor mRNA, which is not protected since IRPs are not bound to it (6Klausner R.D. Rouault T.A. Harford J.B. Cell. 1993; 72: 19-28Abstract Full Text PDF PubMed Scopus (1045) Google Scholar, 7Hentze M.W. Kühn L.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8175-8182Crossref PubMed Scopus (1126) Google Scholar). However, some cells and tissues with specific requirements for iron probably evolved mechanisms that can override the IRE/IRP-dependent control of transferrin receptor expression. Erythroid cells, which are the most avid consumers of iron in the organism, use a transcriptional mechanism to maintain very high transferrin receptor levels (8Ponka P. Blood. 1997; 89: 1-25Crossref PubMed Google Scholar). Transcriptional regulation is probably also involved in the receptor induction when resting cells are activated to proliferate and during T and B lymphocyte activation (2Ponka P. Lok C.N. Int. J. Biochem. Cell Biol. 1999; (in press)PubMed Google Scholar). Although the transcriptional regulation of the transferrin receptor is not fully understood, deletion analysis identified a minimal region of about 100 base pairs upstream from the transcriptional start site that drives both basal as well as serum/mitogenic stimulation of promoter activity (9Owen D. Kühn L.C. EMBO J. 1987; 6: 1287-1293Crossref PubMed Scopus (168) Google Scholar, 10Ouyang Q. Bommakanti M. Miskimins W.K. Mol. Cell. Biol. 1993; 13: 1796-1804Crossref PubMed Scopus (32) Google Scholar). This promoter region also contains elements such as the Ets-binding site and the adjacent AP-1-like sequence, necessary for the transcriptional up-regulation of the transferrin receptor during erythroid differentiation. 2C. N. Lok and P. Ponka, submitted for publication. 2C. N. Lok and P. Ponka, submitted for publication. Recently hypoxia was shown to increase transferrin receptor expression in a hepatoma cell line (11Toth I. Yuan L. Rogers J.T. Boyce H. Bridges K.R. J. Biol. Chem. 1999; 274: 4467-4473Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar) and endothelial cells (12Dore-Duffy P. Balabanov D. Beaumont T. Hritz M.A. Harik S.I. LaManna J.C. Microvasc. Res. 1999; 57: 75-85Crossref PubMed Scopus (37) Google Scholar), and it has been proposed that this increase is due to the hypoxia-enhanced IRE/IRP-1 binding and consequent stabilization of the transferrin receptor message (11Toth I. Yuan L. Rogers J.T. Boyce H. Bridges K.R. J. Biol. Chem. 1999; 274: 4467-4473Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). However, in the course of our studies on the transcriptional control of the transferrin receptor expression, we have noticed that the promoter region of the transferrin receptor gene contains a sequence very similar to the hypoxia response element (HRE) that mediates transcriptional activation by hypoxia-inducible factor-1 (HIF-1). HIF-1 (13Semenza G.L. Wang G.L. Mol. Cell. Biol. 1992; 12: 5447-5454Crossref PubMed Scopus (2137) Google Scholar, 14Wang G.L. Semenza G.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4304-4308Crossref PubMed Scopus (1176) Google Scholar) is a heterodimer (HIF-1α and HIF-1β, the latter being identical to the aryl hydrocarbon nuclear translocator) transcription factor which activates a wide range of genes encoding proteins that represent an important physiological adaptation to hypoxia (15Bunn H.F. Poyton R.O. Physiol. Rev. 1996; 76: 839-885Crossref PubMed Scopus (1037) Google Scholar). These genes include erythropoietin (Epo) (13Semenza G.L. Wang G.L. Mol. Cell. Biol. 1992; 12: 5447-5454Crossref PubMed Scopus (2137) Google Scholar, 17Beck I. Ramirez S. Weinmann R. Caro J. J. Biol. Chem. 1991; 266: 15563-15566Abstract Full Text PDF PubMed Google Scholar), vascular endothelial growth factor (VEGF) (18Levy A.P. Levy N.S. Wegner S. Goldberg M.A. J. Biol. Chem. 1995; 270: 13333-13340Abstract Full Text Full Text PDF PubMed Scopus (875) Google Scholar), several glycolytic enzymes (19Semenza G.L. Roth P.H. Fang H.M. Wang G.L. J. Biol. Chem. 1994; 269: 23757-23763Abstract Full Text PDF PubMed Google Scholar), glucose transporters (19Semenza G.L. Roth P.H. Fang H.M. Wang G.L. J. Biol. Chem. 1994; 269: 23757-23763Abstract Full Text PDF PubMed Google Scholar), inducible nitric-oxide synthase (20Melillo G. Taylor L.S. Brooks A. Musso T. Cox G.W. Varesio L. J. Biol. Chem. 1997; 272: 12236-12243Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar), heme oxygenase-1 (21Lee P.J. Jiang B.H. Chin B.Y. Iyer N.V. Alam J. Semenza G.L. Choi A.M. J. Biol. Chem. 1997; 272: 5375-5381Abstract Full Text Full Text PDF PubMed Scopus (638) Google Scholar), and transferrin (22Rolfs A. Kvietikova I. Gassmann M. Wenger R.H. J. Biol. Chem. 1997; 272: 20055-20062Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar). Hypoxia-induced activation of HIF-1 involves, at least in part, a decrease in oxygen-sensitive degradation of the HIF-1α and an increase in its binding to the HIF-1-binding site (HBS) present in the HRE (23Huang L.E. Gu J. Schau M. Bunn H.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7987-7992Crossref PubMed Scopus (1820) Google Scholar). In this study we demonstrated that the transferrin receptor gene contains a functional HRE that binds HIF-1, which regulates receptor expression under hypoxic conditions. K562 cells were maintained in RPMI medium supplemented with 10% fetal bovine serum and 2 mmglutamine. For hypoxic stimulation, cells were seeded at 5 × 105 cells/ml and put in modular incubator. Hypoxic conditions were produced by purging the incubator with 95% N2, 5% CO2 at 2 pounds/square inch for 15 min, after which the incubator was sealed for 16 h. The partial pressure of O2 of the medium in hypoxic condition was below 22 mm Hg as measured by a blood gas analyzer. After incubation at normal or low O2 tension, the cells were washed, and the total RNA was isolated by guanidine thiocyanate/acid phenol extraction. Total RNA (10 μg) was denatured in formamide/formaldehyde, size-fractionated on formaldehyde/agarose gels, and transferred to nitrocellulose membranes. Hybridization was performed using 32P-random-labeled probes derived from human transferrin receptor cDNA and human β-actin cDNA. The transferrin receptor promoter-luciferase reporter plasmid was constructed by subcloning a polymerase chain reaction fragment of the transferrin receptor promoter region (−118 to +14) into pGL2 basic vector (9Owen D. Kühn L.C. EMBO J. 1987; 6: 1287-1293Crossref PubMed Scopus (168) Google Scholar). Mutations of the putative regulatory elements were generated by a unique site elimination mutagenesis kit (Amersham Pharmacia Biotech) and were confirmed by sequencing. The mutagenesis primers and the sense strands of the oligonucleotides used in electrophoresis mobility shift assays were as follows. TR [HBS]: 5′-CGC GAG CGT ACG TGC CTC AGG-3′ TR [HBSm]: 5′-CGC GAG CGT ACt aGt CTC AGG-3′ EPO [HBS]: 5′-GCC CTA CGT GCT GCC TCG CAT-3′ TR[CRE]: 5′-TCA GGA AGT GAC GCA CAG CCC-3′ TR [CREm]: 5′-TCA GGA AGT cga cCA CAG CCC-3′ For each transfection, 1 × 106 K562 cells were transfected with 1 μg of the reporter plasmid by LipofectAMINE (Life Technologies, Inc.), according to the manufacturer's protocol. After transfection, the cells were incubated under the defined conditions for 16 h. Luciferase and galactosidase activities in the cell extracts were determined by chemoluminescence assay kits (Promega and Tropix) with a luminometer (Bio-Orbit). Transfection efficiencies were monitored by co-transfection of 0.2 μg of SV40-galactosidase reporter. In some experiments, 0.5 μg of expression plasmids of HIF-1α and/or HIF-1β (kindly provided by Jerry Pelletier (37Moffett P. Reece M. Pelletier J. Mol. Cell. Biol. 1997; 17: 4933-4947Crossref PubMed Scopus (82) Google Scholar)) were co-transfected with 2 μg of transferrin receptor reporter. The total amounts of the transfected plasmids were normalized by pGEM plasmids. Nuclear extracts were prepared as described previously (13Semenza G.L. Wang G.L. Mol. Cell. Biol. 1992; 12: 5447-5454Crossref PubMed Scopus (2137) Google Scholar), with the final dialysis step omitted. Lysis of cells was done within 5 min of their removal from the hypoxic chamber. The binding reaction mixtures were pre-mixed in a volume of 18 μl with a final concentration of 5 μg of nuclear proteins, 0.2 μg of poly(dI·dC), 100 mmNaCl, 20 mm Hepes, pH 7.6, 5 mm dithiothreitol, 5% glycerol and incubated for 10 min at room temperature. The32P-labeled oligonucleotide probe (20 fmol, 100,000 cpm in 2 μl) was then added, and the incubation was continued at room temperature for a further 30 min. For competition EMSAs, a 100-fold excess of the cold oligonucleotides were added during the preincubation period. For supershift assays, 1 μg of anti-HIF-1α (Transduction Laboratories) or anti-Jun (Santa Cruz) antibodies was added to the complete binding reaction mixtures, and the incubation was allowed to continue for 30 min on ice. The DNA-protein complexes were resolved on 6% polyacrylamide gels as described elsewhere (13Semenza G.L. Wang G.L. Mol. Cell. Biol. 1992; 12: 5447-5454Crossref PubMed Scopus (2137) Google Scholar). Transferrin receptor mRNA levels in cells incubated under control and hypoxic conditions were measured using Northern blot analysis. Fig. 1 shows that after the exposure of both K562 and HeLa cells to hypoxia for 16 h, the levels of transferrin receptor mRNA increase 2- to 3-fold. By contrast, decreases of β-actin mRNA were observed in cells incubated at low O2 pressure. Because the hypoxic induction of proteins often involves binding of HIF-1 to HRE in their respective genes, we examined whether a consensus HRE was present in the transferrin receptor gene. Indeed, we found that the promoter region of the transferrin receptor gene (Fig.2) contains a sequence highly similar to the HRE found in genes for Epo (13Semenza G.L. Wang G.L. Mol. Cell. Biol. 1992; 12: 5447-5454Crossref PubMed Scopus (2137) Google Scholar, 17Beck I. Ramirez S. Weinmann R. Caro J. J. Biol. Chem. 1991; 266: 15563-15566Abstract Full Text PDF PubMed Google Scholar), VEGF (18Levy A.P. Levy N.S. Wegner S. Goldberg M.A. J. Biol. Chem. 1995; 270: 13333-13340Abstract Full Text Full Text PDF PubMed Scopus (875) Google Scholar), and lactate dehydrogenase A (16Firth J.D. Ebert B.L. Ratcliffe P.J. J. Biol. Chem. 1995; 270: 21021-21027Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar, 19Semenza G.L. Roth P.H. Fang H.M. Wang G.L. J. Biol. Chem. 1994; 269: 23757-23763Abstract Full Text PDF PubMed Google Scholar). Most of the hypoxia response elements exhibit multipartite organization including at least one core HBS and one or two cis-acting elements that are necessary for full inducibility of the gene by hypoxia (reviewed in Ref. 24Semenza G.L. J. Lab. Clin. Med. 1998; 131: 207-214Abstract Full Text PDF PubMed Scopus (185) Google Scholar). As shown in Fig. 2, a HIF-1-binding site with the invariant sequence RCGTG is present not only in known hypoxia-responsible genes but also in the transferrin receptor gene. Seven base pairs of the putative HBS of the transferrin receptor gene (TACGTGC) are identical to the HBS of several hypoxia-regulated genes such as Epo (13Semenza G.L. Wang G.L. Mol. Cell. Biol. 1992; 12: 5447-5454Crossref PubMed Scopus (2137) Google Scholar, 17Beck I. Ramirez S. Weinmann R. Caro J. J. Biol. Chem. 1991; 266: 15563-15566Abstract Full Text PDF PubMed Google Scholar), phosphofructose kinase L (19Semenza G.L. Roth P.H. Fang H.M. Wang G.L. J. Biol. Chem. 1994; 269: 23757-23763Abstract Full Text PDF PubMed Google Scholar), inducible nitric-oxide synthase (20Melillo G. Taylor L.S. Brooks A. Musso T. Cox G.W. Varesio L. J. Biol. Chem. 1997; 272: 12236-12243Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar), and transferrin (22Rolfs A. Kvietikova I. Gassmann M. Wenger R.H. J. Biol. Chem. 1997; 272: 20055-20062Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar). Interestingly, transferrin receptor, Epo (13Semenza G.L. Wang G.L. Mol. Cell. Biol. 1992; 12: 5447-5454Crossref PubMed Scopus (2137) Google Scholar) and VEGF (18Levy A.P. Levy N.S. Wegner S. Goldberg M.A. J. Biol. Chem. 1995; 270: 13333-13340Abstract Full Text Full Text PDF PubMed Scopus (875) Google Scholar) genes have a CACAG sequence located in close proximity to the HBS. In addition, both the HBS in the transferrin receptor gene and the HBS in the lactate dehydrogenase A gene (16Firth J.D. Ebert B.L. Ratcliffe P.J. J. Biol. Chem. 1995; 270: 21021-21027Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar, 26Ebert B.L. Bunn H.F. Mol. Cell. Biol. 1998; 18: 4089-4096Crossref PubMed Scopus (229) Google Scholar) have a nearby cyclic AMP-responsive element (CRE). Fig. 2 also shows that the putative HREs in human and mouse transferrin receptor genes (sequence kindly provided by Nancy Andrews and Joanne Levy) are well conserved. To determine whether the HBS in the transferrin receptor gene is an authentic HBS that can be recognized by HIF-1, electrophoretic mobility shift assays were performed using nuclear extracts prepared from cells incubated under hypoxic and normoxic conditions and the radiolabeled oligonucleotide harboring the putative HBS of the transferrin receptor and its flanking sequence. Fig. 3 shows that this oligomer bound constitutive factors present in both normoxic and hypoxic samples. Importantly, an induced binding activity appeared in nuclear extracts prepared from hypoxic cells (Fig. 3, lane 2), but such a binding activity was virtually undetectable in extracts from the normoxic control (Fig. 3, lane 1). This hypoxia-induced activity represents a specific binding factor since an excess of the unlabeled oligomer completely abolished its binding to the radiolabeled probe (Fig. 3, lane 3). Furthermore, an oligomer harboring the consensus HBS of the Epo gene also effectively competed with the radiolabeled probe for the binding activity (Fig. 3, lane 5). In contrast, the transferrin receptor oligomer whose putative HBS was mutated neither competed with the probe for the binding activity (Fig. 3, lane 4) nor bound hypoxia-induced factors when it was radiolabeled (Fig. 3, lane 10). The identity of the binding activity induced by hypoxia was investigated by supershift assays using antibodies raised against the HIF-1α. As shown in Fig. 3 (lane 6), anti-HIF-1α was able to “supershift” the hypoxia-induced binding activity. Collectively these data indicate that the HBS of the transferrin receptor behaves similarly as consensus HBS and that it can bind HIF-1. To further investigate the function of a potential HRE of the transferrin receptor gene, a reporter gene construct linking the promoter region containing the HRE and a luciferase gene was constructed and transiently transfected into K562 cells (Fig. 4). This promoter region conferred a basal promoter activity under normoxic conditions. Exposure of the cells to hypoxia resulted in an approximately 8-fold increase in promoter activity, but disruption of the putative HBS attenuated the hypoxic stimulation of the transferrin receptor promoter activity by 80%. These data indicate that the HBS in the transferrin receptor promoter is functionally identical to the consensus HBS present in other hypoxia-regulated genes. We also tested whether the CRE site plays a role in response to hypoxia. Although a 7-fold higher activity was still observed in hypoxia-stimulated samples as compared with normoxic samples, the mutation of the CRE significantly reduced both basal and hypoxia-stimulated promoter activity (Fig. 4). Taken together, these results indicate that the HBS is specifically involved in the hypoxia-stimulated promoter activity and that the CRE site may be required for optimal hypoxic response. The functionality of the transferrin receptor HRE was further studied by transient transfection of HIF-1 subunits together with the transferrin receptor HRE-luciferase reporter gene (Fig.5). Transfections with either HIF-1α or HIF-β subunit moderately increased the promoter activity whereas co-transfections with both α and β subunits resulted in a further significant increase in the reporter activity. In contrast, transfection of HIF-1 did not affect the activity of the luciferase reporter when the transferrin receptor HBS was mutated. The biological response induced by hypoxia can be mimicked by treatment with cobaltous ion or iron chelators, which mediate their effects in part by activating HIF-1 (presumably through perturbation of oxygen-sensing processes) (reviewed in Ref. 15Bunn H.F. Poyton R.O. Physiol. Rev. 1996; 76: 839-885Crossref PubMed Scopus (1037) Google Scholar). As shown in Fig.6, treatment of cells with either cobalt chloride or the iron chelator desferrioxamine moderately stimulated transferrin receptor-HRE reporter activity, induced HIF-1 binding to the HBS of the transferrin receptor gene in EMSA and enhanced transferrin receptor mRNA expression. These results further support the notion that the transferrin receptor gene is activated by hypoxia and that it contains functional HRE. This study demonstrated that transferrin receptor gene is hypoxically induced in K562 and HeLa cells (Fig. 1). One possible mechanism by which hypoxia can regulate transferrin receptor mRNA expression is through the transcription factor HIF-1. Most HIF-1-regulated genes contain an enhancer sequence termed HRE, which harbors the HBS as well as one or two additional cis-acting elements in the vicinity of the HBS (reviewed in Ref. 24Semenza G.L. J. Lab. Clin. Med. 1998; 131: 207-214Abstract Full Text PDF PubMed Scopus (185) Google Scholar). Indeed, we demonstrated that the transferrin receptor promoter region contains a functional HRE with a HBS that can bind HIF-1. The HRE of the transferrin receptor exhibits multipartite organization similar to several known HREs of genes such as Epo and VEGF (Fig. 2). The HREs of these genes, as well as that of transferrin receptor gene, contain HBS with the core sequence RCGTG and an adjacent CACAG sequence. The HRE of the transferrin receptor is also similar to that of lactate dehydrogenase A, both of which contain the HBS and a nearby CRE site (Fig. 2). The structural features of the HRE in the transferrin receptor (HBS, CRE, and CACAG sequences) are well conserved in the corresponding mouse sequence (Fig. 2). These sequence data have, therefore, suggested that transferrin receptor HREs have functional importance. We confirmed this by electrophoretic mobility shift assays, which showed that the HBS of the human transferrin receptor gene binds HIF-1 (Fig. 3). Moreover, our studies clearly demonstrated that the wild type but not HBS-mutated transferrin receptor promoter (−118 to +14) conferred hypoxic inducibility to the luciferase reporter gene in K562 cells (Fig. 4). Furthermore, transient co-expression of the two HIF-1 subunits (HIF-1α and HIF-1β) enhanced the transferrin receptor promoter activity (Fig. 5). These results indicate that the transferrin receptor gene contains a functional HRE. In addition, cobaltous ion, which can mimic hypoxia, also stimulated both HIF-1 binding to the HBS of the transferrin receptor and activated its promoter activity (Fig. 6). Interestingly, desferrioxamine had effects very similar to those seen with cobalt (Fig. 6). Desferrioxamine lowers intracellular iron levels and is thought to perturb a redox-based oxygen-sensing process (reviewed in Ref. 15Bunn H.F. Poyton R.O. Physiol. Rev. 1996; 76: 839-885Crossref PubMed Scopus (1037) Google Scholar). It is noteworthy that K562 cells treated with desferrioxamine were demonstrated to have a higher transcription rate of transferrin receptor than iron-replete cells (25Rao K. Harford J.B. Rouault T. McClelland A. Ruddle F.H. Klausner R.D. Mol. Cell. Biol. 1986; 6: 236-240Crossref PubMed Scopus (91) Google Scholar). However, there is no doubt that desferrioxamine increases transferrin receptor mRNA levels via the IRE/IRP mechanisms (6Klausner R.D. Rouault T.A. Harford J.B. Cell. 1993; 72: 19-28Abstract Full Text PDF PubMed Scopus (1045) Google Scholar, 7Hentze M.W. Kühn L.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8175-8182Crossref PubMed Scopus (1126) Google Scholar). The similarity between the regulation of transferrin receptor and lactate dehydrogenase A by HIF-1 is of particular interest (Fig. 2). As already mentioned, the promoter regions of both the transferrin receptor and lactate dehydrogenase A genes contain HRE comprised of a HBS and a nearby CRE site. Although the HBS is clearly necessary for hypoxia-enhanced activity of both promoters, the CRE site is required for optimal hypoxic response. The CRE site of the lactate dehydrogenase A was shown to bind CREB-1/ATF-1 transcription factors (26Ebert B.L. Bunn H.F. Mol. Cell. Biol. 1998; 18: 4089-4096Crossref PubMed Scopus (229) Google Scholar). Interestingly, both HIF-1 and CREB-1/ATF-1 interact at the HRE with p300/CREB-binding protein (26Ebert B.L. Bunn H.F. Mol. Cell. Biol. 1998; 18: 4089-4096Crossref PubMed Scopus (229) Google Scholar, 27Arany Z. Huang L.E. Eckner R. Bhattacharya S. Jiang C. Goldberg M.A. Bunn H.F. Livingston D.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12969-12973Crossref PubMed Scopus (617) Google Scholar), the transcriptional adapter proteins that act as signaling bridges between a number of specific DNA-bound transcription factors and the basal transcription machinery. The CRE site of the transferrin receptor gene also recognizes CREB-1/ATF-1, 3C. N. Lok and P. Ponka, unpublished data. and hence, it seems likely that similar molecular mechanisms are involved in the HIF-1-mediated transcription. It is now well established that post-transcriptional regulation via IRE/IRP system plays an important role in the control of transferrin receptor expression in response to changes in intracellular iron levels (6Klausner R.D. Rouault T.A. Harford J.B. Cell. 1993; 72: 19-28Abstract Full Text PDF PubMed Scopus (1045) Google Scholar, 7Hentze M.W. Kühn L.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8175-8182Crossref PubMed Scopus (1126) Google Scholar). When cellular iron becomes limiting, IRP-2 is present in the cytosol, and IRP-1 is recruited into a high affinity binding state. The binding of IRPs to the IREs in the 3′-untranslated region of transferrin receptor mRNA stabilizes this transcript. On the other hand, when intracellular iron is plentiful, IRP-1 contains a [4Fe-4S] cluster, in which form it is unable to bind to IREs, and IRP-2 is degraded. Hence, in iron-replete cells, IRPs are not available for binding to the IREs, resulting in a rapid degradation of transferrin receptor mRNA. However, iron is not the only species that modulates IRP-1 binding activity, IRP-2 levels, and consequently, transferrin receptor expression. RNA binding activity of IRP-1 can be stimulated by nitric oxide (28Weiss G. Goossen B. Doppler W. Fuchs D. Pantopoulos K. Werner-Felmayer G. Wachter H. Hentze M.W. EMBO J. 1993; 12: 3651-3657Crossref PubMed Scopus (338) Google Scholar, 29Richardson D.R. Neumannova V. Nagy E. Ponka P. Blood. 1995; 86: 3211-3219Crossref PubMed Google Scholar) or hydrogen peroxide (30Pantopoulos K. Hentze M.W. EMBO J. 1995; 14: 2917-2924Crossref PubMed Scopus (292) Google Scholar), suggesting that “oxidative stress” affects cellular iron metabolism. Since cellular production of reactive oxygen species is related to O2 concentration, the effects of hypoxia on IRP-1 and IRP-2 RNA binding activity were investigated, but the results obtained are somewhat controversial. Hanson and Leibold (31Hanson E.S. Leibold E.A. J. Biol. Chem. 1998; 273: 7588-7593Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar) showed that hypoxia inactivated IRP-1 binding to RNA and recently reported that hypoxia increased IRP-2 levels by a post-translational mechanism involving protein stability (32Hanson E.S. Foot L.M. Leibold E.A. J. Biol. Chem. 1999; 274: 5047-5052Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). However, it is unclear what the overall functional consequences of these changes are, since transferrin receptor expression after hypoxia remains to be examined. On the other hand, Toth et al. (11Toth I. Yuan L. Rogers J.T. Boyce H. Bridges K.R. J. Biol. Chem. 1999; 274: 4467-4473Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar) found that hypoxia enhances IRE/IRP-1 binding that was associated with a significant increase in transferrin receptor mRNA levels. Our findings reported here, together with the aforementioned data (11Toth I. Yuan L. Rogers J.T. Boyce H. Bridges K.R. J. Biol. Chem. 1999; 274: 4467-4473Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 31Hanson E.S. Leibold E.A. J. Biol. Chem. 1998; 273: 7588-7593Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 32Hanson E.S. Foot L.M. Leibold E.A. J. Biol. Chem. 1999; 274: 5047-5052Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar), indicate that multiple hypoxia-stimulated mechanisms may exist to modulate transferrin receptor expression. It is not surprising that both transcriptional and post-transcriptional mechanisms operate to regulate transferrin receptor expression, as in the case of prototypic hypoxia-inducible genes such as erythropoietin and VEGF (reviewed in Ref. 15Bunn H.F. Poyton R.O. Physiol. Rev. 1996; 76: 839-885Crossref PubMed Scopus (1037) Google Scholar). Our data indicate the transferrin receptor gene possesses a functional HRE that plays a role in hypoxia-regulated transcription. It is tempting to speculate that the HRE of the transferrin receptor gene, in cooperation with other specific enhancers, is involved in the receptor transcription in a tissue- and stage-specific manner. In addition to transferrin receptor, several other genes involved in iron transport appear to be modulated by low oxygen tension. Transferrin expression in hepatoma cells has been shown to be stimulated by hypoxia via a HIF-1/HRE-mediated mechanism (22Rolfs A. Kvietikova I. Gassmann M. Wenger R.H. J. Biol. Chem. 1997; 272: 20055-20062Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar). Moreover, a putative HBS has been identified in the promoter region of Nramp2/DCT1 (33Lee P.L. Gelbart T. West C. Halloran C. Beutler E. Blood Cells Mol. Dis. 1998; 24: 199-215Crossref PubMed Scopus (271) Google Scholar). Taken together, these reports support a model that hypoxia may augment the overall iron transport machinery, resulting in increasing iron uptake into the cells. Although iron is involved in multiple physiological processes such as oxygen transport, oxidative energy production, and cell growth and development, the biological significance of enhanced transferrin receptor expression during hypoxia remains to be determined. The most typical physiological response elicited by hypoxia is the increase in production of erythropoietin, which stimulates erythropoiesis. Differentiation of erythroid precursors to hemoglobin-synthesizing cells is associated with an enhanced iron uptake mediated by the increase in transferrin receptor expression in the developing erythroid cells. There is no doubt that erythropoietin-mediated mechanisms directly stimulate transferrin receptor expression (8Ponka P. Blood. 1997; 89: 1-25Crossref PubMed Google Scholar), and hence, the effect of hypoxia on receptor expression can be only partial. However, it cannot be ruled out that both erythropoietin and hypoxia can cooperate to drive maximal expression of transferrin receptors under severe hypoxic conditions. Alternatively, it will be interesting to examine the effect of hypoxia on transferrin receptor expression in heart or skeletal muscle as the enhanced synthesis of hemoproteins such as myoglobin in these tissues during hypoxia has been demonstrated (34Terrados N. Jansson E. Sulven C. Kaijser L. J. Appl. Physiol. 1990; 68: 2369-2372Crossref PubMed Scopus (204) Google Scholar, 35Guiang 3rd, S.F. Widness J.A. Flanagan K.B. Schmidt R.L. Radmer W.J. Georgieff M.K. J. Dev. Physiol. (Oxf.). 1993; 19: 99-104PubMed Google Scholar, 36Guiang 3rd, S.F. Merchant J.R. Eaton M.A. Fandel K.B. Georgieff M.K. Can. J. Physiol. Pharmacol. 1998; 76: 930-936Crossref PubMed Google Scholar). We are grateful to Lukas Kühn for human transferrin receptor plasmids and Jerry Pelletier and Peter Moffett for HIF-1 plasmids. We are also grateful to Nancy Andrews and Joanne Levy for mouse transferrin receptor gene sequences. We thank Joan Buss, Sangwon Kim, and Emanuel Necas for useful comments, Eva Nagy for technical assistance, and Sandy Fraiberg for editorial assistance." @default.
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