Subcellular localization of HIF-1(alpha) and...

10
INTRODUCTION In response to reduced oxygenation, activation of the hypoxia- inducible factor-1 (HIF-1) regulates transcription of several genes involved in oxygen homeostasis (reviewed in Bunn and Poyton, 1996; Semenza, 1998; Wenger and Gassmann, 1997). The transcription factor HIF-1 was originally discovered as a critical factor binding to the hypoxia-inducible 3enhancer of the erythropoietin (Epo) gene. Subsequently, HIF-1 was shown to be involved in oxygen-dependent expression of many other genes, including vascular endothelial growth factor (VEGF), glycolytic enzymes, glucose transporter-1 (Glut-1), transferrin, inducible nitric oxide synthase and heme oxygenase-1 (Wenger and Gassmann, 1997). HIF-1 is a heterodimeric complex composed of the two basic-helix-loop-helix (bHLH) Per- ARNT-Sim (PAS) subunits HIF-1α and HIF-1β (Wang et al., 1995a). As determined in HeLa cells, highest HIF-1α protein levels are reached at 0.5% oxygen (Jiang et al., 1996b) by a process that involves redox-dependent proteolytic stabilization to prevent HIF-1α ubiquitinylation and rapid degradation in proteasomes (Huang et al., 1998, 1996; Salceda and Caro, 1997). HIF-1β is identical to the aryl hydrocarbon receptor nuclear translocator (ARNT), which was first cloned as a heterodimerization partner of the aryl hydrocarbon receptor (AhR), also known as the dioxin receptor (reviewed in Gassmann and Wenger, 1997; Hankinson, 1995; Schmidt and Bradfield, 1996). By using an ARNT-mutant cell line (Hepa1C4) derived from Hepa1 mouse hepatoma cells, we (Gassmann et al., 1997; Gradin et al., 1996) and others (Forsythe et al., 1996; Salceda et al., 1996; Wood et al., 1996) have shown that ARNT is indispensable for HIF-1 DNA binding and transactivation. Mice homozygous for a targeted deletion in the gene encoding HIF-1α are not viable and die around midgestation, mainly due to defective vascularization, heart malformations and failure in neuronal tube closure (Iyer et al., 1998). In addition, embryonic stem (ES) cell-derived solid tumour formation is also affected in the absence of HIF-1α (Carmeliet et al., 1998; Ryan et al., 1998). Similar to HIF-1α, ARNT 1203 Journal of Cell Science 112, 1203-1212 (1999) Printed in Great Britain © The Company of Biologists Limited 1999 JCS0249 Hypoxia-inducible factor-1 (HIF-1) is a master regulator of mammalian oxygen homeostasis. HIF-1 consists of two subunits, HIF-1α and the aryl hydrocarbon receptor nuclear translocator (ARNT). Whereas hypoxia prevents proteasomal degradation of HIF-1α, ARNT expression is thought to be oxygen-independent. We and others previously showed that ARNT is indispensable for HIF-1 DNA-binding and transactivation function. Here, we have used ARNT-mutant mouse hepatoma and embryonic stem cells to examine the requirement of ARNT for accumulation and nuclear translocation of HIF-1α in hypoxia. As shown by immunofluorescence, HIF-1α accumulation in the nucleus of hypoxic cells was independent of the presence of ARNT, suggesting that nuclear translocation is intrinsic to HIF-1α. Co- immunoprecipitation of HIF-1α together with ARNT could be performed in nuclear extracts but not in cytosolic fractions, implying that formation of the HIF-1 complex occurs in the nucleus. A proteasome inhibitor and a thiol- reducing agent could mimic hypoxia by inducing HIF-1α in the nucleus, indicating that escape from proteolytic degradation is sufficient for accumulation and nuclear translocation of HIF-1α. During biochemical separation, both HIF-1α and ARNT tend to leak from the nuclei in the absence of either subunit, suggesting that heterodimerization is required for stable association within the nuclear compartment. Nuclear stabilization of the heterodimer might also explain the hypoxically increased total cellular ARNT levels observed in some of the cell lines examined. Key words: Dioxin receptor, Erythropoietin, Gene expression, Oxygen, Protein stability SUMMARY Induction and nuclear translocation of hypoxia-inducible factor-1 (HIF-1): heterodimerization with ARNT is not necessary for nuclear accumulation of HIF-1α Dmitri Chilov 1 , Gieri Camenisch 1 , Ivica Kvietikova 1, *, Urs Ziegler 2 , Max Gassmann 1 and Roland H. Wenger 1,‡ 1 Institute of Physiology and 2 Institute of Anatomy, University of Zürich-Irchel, CH-8057 Zürich, Switzerland *Present address: Institut de Biochimie, Université de Lausanne, Ch. des Boveresses 155, CH-1066 Epalinges, Switzerland Author for correspondence (e-mail: [email protected]) Accepted 10 February; published on WWW 23 March 1999

Transcript of Subcellular localization of HIF-1(alpha) and...

Page 1: Subcellular localization of HIF-1(alpha) and ARNTjcs.biologists.org/content/joces/112/8/1203.full.pdf · HIF-1βis identical to the aryl hydrocarbon receptor nuclear translocator

INTRODUCTION

In response to reduced oxygenation, activation of the hypoxia-inducible factor-1 (HIF-1) regulates transcription of severalgenes involved in oxygen homeostasis (reviewed in Bunn andPoyton, 1996; Semenza, 1998; Wenger and Gassmann, 1997).The transcription factor HIF-1 was originally discovered as acritical factor binding to the hypoxia-inducible 3′ enhancer ofthe erythropoietin (Epo) gene. Subsequently, HIF-1 was shownto be involved in oxygen-dependent expression of many othergenes, including vascular endothelial growth factor (VEGF),glycolytic enzymes, glucose transporter-1 (Glut-1), transferrin,inducible nitric oxide synthase and heme oxygenase-1 (Wengerand Gassmann, 1997). HIF-1 is a heterodimeric complexcomposed of the two basic-helix-loop-helix (bHLH) Per-ARNT-Sim (PAS) subunits HIF-1α and HIF-1β (Wang et al.,1995a). As determined in HeLa cells, highest HIF-1α proteinlevels are reached at 0.5% oxygen (Jiang et al., 1996b) by aprocess that involves redox-dependent proteolytic stabilizationto prevent HIF-1α ubiquitinylation and rapid degradation in

proteasomes (Huang et al., 1998, 1996; Salceda and Caro,1997).

HIF-1β is identical to the aryl hydrocarbon receptor nucleartranslocator (ARNT), which was first cloned as aheterodimerization partner of the aryl hydrocarbon receptor(AhR), also known as the dioxin receptor (reviewed inGassmann and Wenger, 1997; Hankinson, 1995; Schmidt andBradfield, 1996). By using an ARNT-mutant cell line(Hepa1C4) derived from Hepa1 mouse hepatoma cells, we(Gassmann et al., 1997; Gradin et al., 1996) and others(Forsythe et al., 1996; Salceda et al., 1996; Wood et al., 1996)have shown that ARNT is indispensable for HIF-1 DNAbinding and transactivation.

Mice homozygous for a targeted deletion in the geneencoding HIF-1α are not viable and die around midgestation,mainly due to defective vascularization, heart malformationsand failure in neuronal tube closure (Iyer et al., 1998). Inaddition, embryonic stem (ES) cell-derived solid tumourformation is also affected in the absence of HIF-1α (Carmelietet al., 1998; Ryan et al., 1998). Similar to HIF-1α, ARNT

1203Journal of Cell Science 112, 1203-1212 (1999)Printed in Great Britain © The Company of Biologists Limited 1999JCS0249

Hypoxia-inducible factor-1 (HIF-1) is a master regulator ofmammalian oxygen homeostasis. HIF-1 consists of twosubunits, HIF-1α and the aryl hydrocarbon receptornuclear translocator (ARNT). Whereas hypoxia preventsproteasomal degradation of HIF-1α, ARNT expression isthought to be oxygen-independent. We and otherspreviously showed that ARNT is indispensable for HIF-1DNA-binding and transactivation function. Here, we haveused ARNT-mutant mouse hepatoma and embryonic stemcells to examine the requirement of ARNT foraccumulation and nuclear translocation of HIF-1α inhypoxia. As shown by immunofluorescence, HIF-1αaccumulation in the nucleus of hypoxic cells wasindependent of the presence of ARNT, suggesting thatnuclear translocation is intrinsic to HIF-1α. Co-immunoprecipitation of HIF-1 α together with ARNT couldbe performed in nuclear extracts but not in cytosolic

fractions, implying that formation of the HIF-1 complexoccurs in the nucleus. A proteasome inhibitor and a thiol-reducing agent could mimic hypoxia by inducing HIF-1αin the nucleus, indicating that escape from proteolyticdegradation is sufficient for accumulation and nucleartranslocation of HIF-1α. During biochemical separation,both HIF-1α and ARNT tend to leak from the nuclei in theabsence of either subunit, suggesting thatheterodimerization is required for stable association withinthe nuclear compartment. Nuclear stabilization of theheterodimer might also explain the hypoxically increasedtotal cellular ARNT levels observed in some of the cell linesexamined.

Key words: Dioxin receptor, Erythropoietin, Gene expression,Oxygen, Protein stability

SUMMARY

Induction and nuclear translocation of hypoxia-inducible factor-1 (HIF-1):

heterodimerization with ARNT is not necessary for nuclear accumulation of

HIF-1α

Dmitri Chilov 1, Gieri Camenisch 1, Ivica Kvietikova 1,*, Urs Ziegler 2, Max Gassmann 1 and Roland H. Wenger 1,‡

1Institute of Physiology and 2Institute of Anatomy, University of Zürich-Irchel, CH-8057 Zürich, Switzerland*Present address: Institut de Biochimie, Université de Lausanne, Ch. des Boveresses 155, CH-1066 Epalinges, Switzerland‡Author for correspondence (e-mail: [email protected])

Accepted 10 February; published on WWW 23 March 1999

Page 2: Subcellular localization of HIF-1(alpha) and ARNTjcs.biologists.org/content/joces/112/8/1203.full.pdf · HIF-1βis identical to the aryl hydrocarbon receptor nuclear translocator

1204

deficiency is also embryonic lethal (Kozak et al., 1997;Maltepe et al., 1997), indicating that the heterodimeric HIF-1complex is a non-redundant master regulator of oxygenhomeostasis. Interestingly, targeted deletion of HIF-2α alsoresults in embryonic lethality, because the impaired function ofthe organ of Zuckerkandl results in reduced catecholaminesynthesis (Tian et al., 1998).

HIF-1α protein stabilization in hypoxia is generallyconsidered to be the rate-limiting step in HIF-1 activation.However, a (weaker) concomitant increase in ARNT proteinlevels has also been observed by immunoblot analyses ofnuclear extracts derived from many different cell types (Iyer etal., 1998; Jiang et al., 1997a,b, 1996a,b; Lee et al., 1997; Liuet al., 1998; Martin et al., 1998; Wang et al., 1995a,b,c). Thesignificance of these observations has not been furtherinvestigated, but hypoxically increased ARNT levels in nuclearextracts might be related to HIF-1α nuclear translocation. Toclarify the role of ARNT in HIF-1α regulation, we analyzedHIF-1α and ARNT levels and subcellular localizationfollowing exposure to hypoxia in cell lines that are either wildtype or deficient for ARNT.

MATERIALS AND METHODS

Cell culture and hypoxic inductionThe human HeLa epitheloid carcinoma and Hep3B hepatoma celllines were obtained from American Type Culture Collection (ATCCnumbers CCL-2 and HB-8064, respectively). The human LN229glioblastoma cell line (Wenger et al., 1997) was a kind gift of E. G.Van Meir (Lausanne, Switzerland). The mouse L929 fibroblast (ATCCCCL-1), and the Hepa1 (also termed Hepa1c1c7), Hepa1C4 andVT{2} hepatoma cell lines, originally developed by O. Hankinson andco-workers (Hoffman et al., 1991), were kindly provided by V.O’Donnall (Bern, Switzerland), L. Poellinger (Stockholm, Sweden)and J. Caro (Philadelphia, PA), respectively. Wild-type and ARNT-deficient (Maltepe et al., 1997) R1 ES cells were kind gifts of C.Simon (Chicago, IL). All cells were cultured in DMEM medium (highglucose, Gibco-BRL) supplemented with 10% heat-inactivated fetalcalf serum (FCS, Boehringer-Mannheim), 100 i.u./ml penicillin, 100µg/ml streptomycin, 1× MEM non-essential amino acids, 2 mM L-glutamine and 1 mM sodium pyruvate (all Gibco-BRL) in ahumidified atmosphere containing 5% CO2 at 37°C. Oxygen tensionsin the incubator (Forma Scientific, model 3319) were either 140 mmHg (20% O2 v/v, normoxia) or 7 mm Hg (1% O2 v/v, hypoxia). Thecells were exposed to hypoxia for 4 hours. Where indicated,lactacystin (10 mM in DMSO, Calbiochem) or N-(2-mercaptopropionyl)-glycine (NMPG, 1 M in H2O, Fluka) was addedto normoxic cells.

Protein extractionsNuclear extracts and cytoplasmic fractions were prepared as describedpreviously (Kvietikova et al., 1995). Briefly, 1×108 cells were washedtwice, collected in ice-cold PBS and pelleted. After incubation inbuffer A (10 mM Tris-HCl, pH 7.8, 1.5 mM MgCl2, 10 mM KCl) onice for 10 minutes, the cells were lysed by 10 strokes of a Douncehomogenizer and centrifuged for 10 minutes at 1500 g. Cytoplasmicfractions were obtained by re-centrifugation of the supernatant for 10minutes at 20,000 g. The pelleted nuclei were extracted with buffer C(420 mM KCl, 20 mM Tris-HCl, pH 7.8, 1.5 mM MgCl2, 20%glycerol) at 4°C for 30 minutes with gentle agitation. Immediatelybefore use, buffers A and C were supplemented with 0.5 mMdithiothreitol (DTT), 1 mM Na3VO4, and a protease inhibitor cocktailconsisting of 0.4 mM phenylmethylsulfonyl fluoride and 2 µg/ml each

of leupeptin, pepstatin and aprotinin (all obtained from Sigma). Thenuclear extract was centrifuged and the supernatant was dialyzedtwice against buffer D (20 mM Tris-HCl, pH 7.8, 100 mM KCl, 0.2mM EDTA, 20% glycerol). For total cellular lysates, the culturemedium was removed, the cells were washed twice with ice-cold PBSand incubated in lysis buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA,150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1%SDS plus protease inhibitor cocktail) for 10 minutes on ice. Thelysates were sonicated and centrifuged at 700 g for 20 minutes. Thesupernatant was collected and stored frozen at −80°C. Proteinconcentrations were determined by the Bradford protein assay (Bio-Rad) or the BCA assay (Pierce) using bovine serum albumin as astandard.

Immunoblot analysisProtein extracts were electrophoresed through SDS-polyacrylamidegels and transferred to nitrocellulose membranes (Schleicher andSchuell) using standard procedures. Equal protein loading andtransfer was verified by Ponceau staining (Sigma). The membranewas blocked with 4% (w/v) instant non-fat milk powder andincubated for 2 hours with the indicated antibodies diluted in PBScontaining 4% milk powder. An anti-HIF-1α chicken polyclonal IgYand a mouse monoclonal IgG1 antibody (designated mgc3) wereraised against a bacterially expressed GST-HIF-1α fusion proteincontaining amino acids 530-825 of human HIF-1α. The generationand purification of these antibodies are described in more detailelsewhere (Camenisch et al., 1999). Rabbit anti-ARNT polyclonalIgG antibodies and the mouse anti-ARNT monoclonal antibody2B10 (Hord and Perdew, 1994) were kindly provided by L.Poellinger and G. H. Perdew (Pennsylvania, PA) or purchased fromAffinity BioReagents. Rabbit-anti Sp1 polyclonal IgG antibodieswere purchased from SantaCruz Biotechnology. The respectivehorseradish peroxidase-coupled secondary antibodies were: rabbitanti-chicken (Promega), goat anti-mouse (Pierce) or goat anti-rabbit(Sigma), and Super Signal Chemiluminescent Substrate (Pierce) wasused for their detection.

ImmunoprecipitationNuclear extracts (200 µg) and cytoplasmic fractions (2 mg) frommouse hepatoma cell lines were incubated with the affinity-purifiedanti-HIF-1α IgY antibody (30 ng) overnight on ice followed byincubation with a rabbit anti-chicken antibody (Promega), or with arabbit anti-ARNT antibody. Protein A-Sepharose (Pharmacia) wasadded and incubated with rotation for 30 minutes at 4°C. Followingcentrifugation of the precipitate at 15,000 g, the pellet was washedthree times with either buffer A or buffer C (see above) forcytoplasmic and nuclear fractions, respectively, and twice with 10 mMTris-HCl, pH 7.5. Finally, the precipitates were analyzed byimmunoblotting using either the anti-HIF-1α IgY or the anti-ARNT2B10 antibody.

Electrophoretic mobility shift assay (EMSA)An oligonucleotide probe derived from the Epo 3′ enhancer waspurified and labelled as described previously (Kvietikova et al., 1995).DNA-protein binding reactions were carried out for 20 minutes at 4°Cin 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 mMKCl, 50 mM NaCl, 1 mM MgCl2, 1 mM EDTA, 5 mM DTT and 5%glycerol and run on 4% non-denaturating 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 wereautoradiographed. In vitro transcription and translation of ARNT wereperformed using the plasmid pBSArntKS+ (Hoffman et al., 1991) andthe TNT® coupled transcription-translation rabbit reticulocyte lysatesystem (Promega) under conditions recommended by themanufacturers. For supershift analysis, 1 µl of anti-HIF-1α mgc3

D. Chilov and others

Page 3: Subcellular localization of HIF-1(alpha) and ARNTjcs.biologists.org/content/joces/112/8/1203.full.pdf · HIF-1βis identical to the aryl hydrocarbon receptor nuclear translocator

1205Subcellular localization of HIF-1α and ARNT

hybridoma supernatant was added to the completed EMSA reactionmixture and incubated for 16 hours at 4°C prior to loading.

Indirect immunofluorescence microscopyAdherent cells were fixed with 4% formaldehyde in PBS (pH 8.0) for10 minutes, washed three times with PBS, permeabilized with 0.5%Triton X-100 in PBS for 5 minutes and rinsed again three times withPBS (all at room temperature). After blocking nonspecific binding byadding 10% FCS in PBS for 30 minutes, the cells were incubated witheither the affinity-purified anti-HIF-1α IgY (1-2 ng/µl in PBS) or themonoclonal anti-ARNT 2B10 antibody at 37°C for 1 hour. Primaryantibodies were detected by incubation with either an FITC-conjugated rabbit anti-chicken antibody (Promega) or Cy3-conjugateddonkey anti-mouse antibody (Jackson Immunoresearch Laboratory) atroom temperature for 30 minutes. Following extensive washing inPBS and mounting in Dabco solution (Sigma), cells were analyzed byconfocal laser scanning microscopy (CLSM-310, Zeiss). For antibodydepletion experiments, the IgY antibody was incubated with a 100-fold excess of GST-HIF-1α fusion protein for 30 minutes on ice. Thesolution was centrifuged at 10,000 g for 30 minutes and thesupernatant was used to stain the cells as described above.

RESULTS

ARNT is induced in nuclear extracts but not in totalcellular lysates from cells exposed to hypoxiaHIF-1α and ARNT protein levels were examined incytoplasmic fractions and nuclear extracts derived from HeLa,Hep3B, LN229, L929 and Hepa1 cells that were cultured ateither normoxia or hypoxia. The samples were analyzed byimmunoblotting using an affinity-purified chicken anti-HIF-1αIgY or a rabbit anti-ARNT antibody. As shown in Fig. 1A,

HIF-1α protein was detected exclusively in nuclear extracts ofhypoxically induced cells. No additional bands were observed,confirming the specificity of the IgY antibody (not shown).While ARNT was not detectable in normoxic nuclear extractsof HeLa, Hep3B and Hepa1 cells, it was found in LN229 andL929 cells. Unexpectedly, following exposure to hypoxia,increased ARNT protein levels were found in nuclear extractsof all cell lines. No HIF-1α and ARNT could be detected inthe same amount of cytoplasmic protein derived from the samecell lines (Fig. 1A). We next tested whether nuclear ARNTaccumulation in hypoxia is due to increased cellular ARNTprotein concentrations. As shown in Fig. 1B, ARNT levels intotal cellular lysates remained unchanged after hypoxicinduction of the human cell lines HeLa, Hep3B and LN229,whereas ARNT was repeatedly induced in the mouse L929 andHepa1 cell lines. HIF-1α was not detectable in normoxic totalcellular lysates (not shown).

Taken together, these results might imply that hypoxic nuclearARNT accumulation was a result of translocation from thecytoplasm to the nucleus. At least in some cell lines, increasedARNT production and/or reduced degradation also contributesto the elevated ARNT levels in nuclear extracts. However, thefact that no cytoplasmic ARNT could be detected in normoxia

Fig. 1. Immunoblot analysis of HIF-1α and ARNT expression insubcellular fractions of various human and mouse cell lines. (A) Thecell lines were cultured under normoxic (20% O2) or hypoxic (1%O2) conditions for 4 hours, and 25 µg of protein from nuclearextracts (n) or cytoplasmic fractions (c) were separated by SDS-PAGE followed by immunoblotting and detection with the polyclonalanti-HIF-1α IgY antibody (upper panel) or the rabbit anti-ARNTantibody (lower panel). (B) ARNT expression analyzed in totalcellular lysates (50 µg). Ponceau staining of the membrane priorincubation with antibodies confirmed equal loading and blottingefficiency.

Fig. 2. Immunoblot analysis of HIF-1α and ARNT expression inwild-type and ARNT-mutant mouse hepatoma cells. (A) Wild-typeHepa-1 and ARNT-mutant Hepa1C4 cells were exposed for 4 hoursto hypoxic conditions, and 50 µg each of nuclear extracts (n) andcytoplasmic fractions (c) were analyzed by immunoblotting. Notethat the amount of loaded protein was increased twofold compared toFig. 1, allowing the detection of cytoplasmic HIF-1α. Representativeresults from a single immunoblot are shown. (B) Hepa1 andHepa1C4 cells were exposed to hypoxia, and the nuclei extractedwith increasing NaCl concentrations. Following incubation with theanti-HIF-1α antibody, the membrane was incubated with an anti-Sp1control antibody. Ponceau staining of the membranes prior toincubation with antibodies confirmed equal loading and blottingefficiency.

Page 4: Subcellular localization of HIF-1(alpha) and ARNTjcs.biologists.org/content/joces/112/8/1203.full.pdf · HIF-1βis identical to the aryl hydrocarbon receptor nuclear translocator

1206

provides evidence against the ‘translocation hypothesis’. Thus,we assume that nuclear ARNT was lost during isolation (butbefore extraction) of the nuclei rather than being induced byhypoxia. This would suggest a higher affinity of ARNT for thenuclear compartment in hypoxia (i.e. in the presence of HIF-1α)than in normoxia (i.e. in the absence of HIF-1α).

Nuclear HIF-1 α accumulation in an ARNT-mutanthepatoma cell lineTo analyze whether ARNT is necessary for hypoxic induction

and nuclear accumulation of HIF-1α, we used an ARNT-mutant Hepa1-derived subline (termed Hepa1C4) that wasoriginally selected for its resistance against 3,4-benzopyrenetreatment (Hoffman et al., 1991). We and others previouslyshowed that in Hepa1C4 cells (1) ARNT mRNA levels weredecreased, (2) hypoxic response of endogenous as well asreporter gene transcription was impaired and (3) no functionalHIF-1 DNA-binding activity was detected (Forsythe et al.,1996; Gassmann et al., 1997; Gradin et al., 1996; Salceda etal., 1996; Wenger et al., 1998; Wood et al., 1996). As shownin Fig. 2A, ARNT protein was also hardly detectable innuclear extracts from normoxic and hypoxic Hepa1C4 cells.Interestingly, HIF-1α was still induced in nuclear extractsfrom Hepa1C4 cells, although at lower levels compared towild-type Hepa1 cells. Reduced HIF-1α levels in nuclearextracts from Hepa1C4 cells were paralleled by increasedHIF-1α levels in the cytoplasmic fraction (Fig. 2A). As forARNT, these findings suggest that the affinity of HIF-1α forthe nuclear compartment is higher in the presence of itsheterodimerization partner. To directly assess this hypothesis,a titration experiment was performed, using increasingconcentrations of NaCl to extract the nuclei. As shown in Fig.2B, at least 300 mM NaCl was necessary to extract HIF-1αfrom wild-type cells, whereas in ARNT-mutant cells, HIF-1αhad already leaked out of the nuclei in the absence of NaCl.In addition, HIF-1α was more efficiently extracted inHepa1C4 than in Hepa1 cells with all the NaCl concentrationsused. Correspondingly, the HIF-1α content was much lowerin cytoplasmic fractions from Hepa1 than from Hepa1C4cells. In contrast, the unrelated transcription factor Sp-1 wasefficiently extracted at 300 mM NaCl in both cell lines. Takentogether, these results suggest that ARNT is required to retainHIF-1α in the nucleus but not for HIF-1α nucleartranslocation.

D. Chilov and others

Fig. 3.Electrophoretic mobility shift assay (EMSA) of nuclearextracts derived from wild-type and ARNT-mutant mouse hepatomacells. Nuclear fractions were prepared from normoxic or hypoxicwild-type Hepa1 and ARNT-mutant Hepa1C4 cells. Nuclear proteinextracts (5 µg) were incubated with a 32P-labelled oligonucleotideprobe carrying the HIF-1 binding site derived from the Epo 3′enhancer. The HIF-1 complex is supershifted by the monoclonal anti-HIF-1α antiboy mgc3. HIF-1 DNA-binding activity in hypoxicARNT-mutant Hepa1C4 nuclear extracts is reconstituted followingaddition of in vitro translated wild-type ARNT protein. Migrationpositions of hypoxia-inducible (HIF-1), constitutive (ATF-1/CREB-1family members) and nonspecific factors are indicated.

Fig. 4. Co-immunoprecipitation of HIF-1α:ARNT complexes from nuclearextracts of hypoxic hepatoma cells.Nuclear extracts (n, 200 µg) andcytoplasmic fractions (c, 2 mg) wereprepared from normoxic and hypoxicwild-type Hepa1 and ARNT-mutantHepa1C4 cells. Following incubationwith either the anti-HIF-1α IgYantibody, followed by a rabbit anti-IgYantibody or the rabbit anti-ARNTantibody, the immune complexes wereprecipitated by addition of protein A sepharose. The precipitates were analyzed by immunoblotting using the monoclonal anti-ARNT2B10 antibody or the polyclonal anti-HIF-1α IgY antibody.

Page 5: Subcellular localization of HIF-1(alpha) and ARNTjcs.biologists.org/content/joces/112/8/1203.full.pdf · HIF-1βis identical to the aryl hydrocarbon receptor nuclear translocator

1207Subcellular localization of HIF-1α and ARNT

Reconstitution of HIF-1 DNA-binding activity innuclear extracts from Hepa1C4 cellsTo prove that nuclear HIF-1α from Hepa1C4 cells is capableof forming a functional complex with ARNT, EMSAs wereperformed using an Epo 3′ enhancer-derived HIF-1 DNA-binding oligonucleotide (Kvietikova et al., 1995). Hepa1C4nuclear extracts were complemented with exogenous ARNTprotein that was translated in vitro using reticulocyte lysates.As shown in Fig. 3, synthetic ARNT could reconstitute HIF-1DNA-binding despite a general inhibitory effect of thereticulocyte lysate on DNA-binding activity of HIF-1 and ATF-1/CREB-1 family members, which assemble the constitutivefactor (Kvietikova et al., 1995). Supershift experiments usingthe monoclonal anti-HIF-1α antibody mgc3 confirmed theidentity of the HIF-1 band (Fig. 3). Unprogrammedreticulocyte lysate lacking HIF-1α and ARNT cDNAs did notyield any detectable bands (not shown).

Formation of the HIF-1 heterodimer in the nucleusTo assess the site of HIF-1 heterodimer formation, co-immunoprecipitation experiments were performed using theaffinity-purified chicken anti-HIF-1α IgY or the rabbit anti-ARNT antibody. Since chicken IgY antibodies have a very lowaffinity for protein A or G, a rabbit anti-IgY antibody was usedas an intermediary for the precipitation with protein A-sepharose beads. The precipitates were analyzed byimmunoblotting using either the chicken anti-HIF-1α IgY or themouse anti-ARNT 2B10 antibody. As shown in Fig. 4 (left),ARNT was co-immunoprecipitated together with HIF-1α from200 µg hypoxic nuclear extract but not from 2 mg cytoplasmicfraction of Hepa1 cells. On the other hand, HIF-1α was co-immunoprecipitated together with ARNT from 200 µg hypoxicnuclear extract but not from 2 mg cytoplasmic fraction of Hepa1cells (Fig. 4, right). Since this represents 40-fold more proteinthan minimally required to detect cytoplasmic HIF-1α in an

Fig. 5. Indirectimmunofluorescence analysis ofHIF-1α expression in normoxic andhypoxic human and mouse celllines. The various cell lines werecultured under normoxic or hypoxicconditions for 4 hours, and preparedfor immunofluorescence asdescribed in Materials and methods.The cells were then incubated withthe affinity-purified anti-HIF-1αIgY antibody followed by a FITC-conjugated rabbit anti-chickenantibody and analyzed by CLSM(HeLa: b,c; Hep3B: f,g; Hepa1: j,k;Hepa1C4: n,o; VT{2}: r,s). Thecorresponding transmission imagesare shown in the outer columns.Normoxic and hypoxic Hepa1 cellswere stained with either the anti-HIF-1α IgY (j,k), or with the IgYantibody that had been depleted ofspecific anti-HIF-1α antibodies bypre-incubation with GST-HIF-1αfusion protein (v,w).

Page 6: Subcellular localization of HIF-1(alpha) and ARNTjcs.biologists.org/content/joces/112/8/1203.full.pdf · HIF-1βis identical to the aryl hydrocarbon receptor nuclear translocator

1208

immunoblot (see Fig. 2A), these results suggest that HIF-1α:ARNT heterodimer formation occurs mainly in the nucleus.No co-immunoprecipitations were obtained from normoxicHepa1 or normoxic and hypoxic Hepa1C4 extracts.

Subcellular localization of HIF-1 α in wild-type andARNT-mutant hepatoma cellsSince HIF-1α was prone to leak from the nuclei duringbiochemical separation in the absence of ARNT (see above),we examined the subcellular localization of HIF-1α byindirect immunofluorescence and confocal laser scanningmicroscopy (CLSM). Normoxic and hypoxic HeLa, Hep3B,wild-type Hepa1, ARNT-mutant Hepa1C4 as well as ARNT-reconstituted VT{2} (Hoffman et al., 1991) cells wereanalyzed using the chicken anti-HIF-1α IgY antibody. Innormoxically cultured cells, very weak immunofluorescencewas detected in the cytoplasm and nuclei of HeLa andHep3B cells (Fig. 5b,f), but not in the other cell lines (Fig.5j,n,r). Upon hypoxic exposure, all cell lines, includingARNT-mutant Hepa1C4 cells, responded with a drasticincrease in HIF-1α levels in the nucleus but not in thenucleoli (Fig. 5c,g,k,o,s), demonstrating that ARNT is notrequired for hypoxic accumulation and nuclear translocationof HIF-1α. In contrast to the immunoblot results (Fig. 2A),HIF-1α was not detectable in the cytoplasm of hypoxicHepa1C4 cells (Fig. 5o), supporting our notion that thepresence of HIF-1α in cytoplasmic fractions is mainly dueto leakage from the nucleus. In human HeLa and Hep3Bcells, the cytoplasmic fluorescence was brighter than that ofmouse Hepa1 cells. To ensure that cytoplasmic staining inHeLa and Hep3B cells was not an experimental artifact,

HeLa cells were stained using the monoclonal anti-HIF-1αantibody mgc3, and a similar cytoplasmic fluorescence wasobserved (data not shown). To confirm the specificity of thechicken anti-HIF-1α IgY antibody, it was pre-absorbed withrecombinant GST-HIF-1α fusion protein and removed bycentrifugation. As demonstrated in Fig. 5w, this treatmentcompletely abolished binding of the antibody to hypoxicHepa1 cells.

Induction and subcellular localization of ARNT inhypoxiaIt has previously been reported that, unlike the dioxin receptor,ARNT levels remain spatially and temporarily constant in thenucleus following treatment with dioxin-analogs (Pollenz,1996). In contrast, in our immunoblotting experiments (seeFigs 1A, 2A), as well as in those of others (see Introduction),ARNT seemed to be regulated by hypoxia. Because these datamight be compromised by artifacts occurring duringbiochemical separation, we performed indirectimmunofluorescence experiments using the anti-ARNTmonoclonal antibody 2B10. As shown in Fig. 6b,c, the ARNTlevel in the nucleus of Hepa1 cells increased followingexposure to hypoxia. A nonspecific cross-reactivity of the2B10 antibody gave rise to background signals in Hepa1 andHepa1C4 cells, which overwhelmed normoxic ARNT signalsin Hepa1 cells. Opposite to Hepa1 cells, no hypoxic ARNTinduction was observed in HeLa cells (Fig. 6j,k). These resultsconfirm our immunoblot data using total cellular extracts (Fig.1B), and show that (1) ARNT is a nuclear protein and itsdisappearance from normoxic nuclear extracts is due to leakageduring preparation of the nuclei, and (2) certain cell lines are

D. Chilov and others

Fig. 6. Indirect immunofluorescence analysis of ARNT expression in normoxic and hypoxic mouse and human cell lines. Wild-type Hepa1(b,c), ARNT-mutant Hepa1C4 (f,g) and HeLa cells (j,k) were incubated with the monoclonal anti-ARNT antibody 2B10 followed by a Cy3-conjugated donkey anti-mouse antibody. The corresponding transmission images are shown in the outer columns.

Page 7: Subcellular localization of HIF-1(alpha) and ARNTjcs.biologists.org/content/joces/112/8/1203.full.pdf · HIF-1βis identical to the aryl hydrocarbon receptor nuclear translocator

1209Subcellular localization of HIF-1α and ARNT

capable of inducing ARNT protein levels in a hypoxia-dependent manner.

Subcellular localization of HIF-1 α in wild-type andARNT-deficient embryonic stem cellsAn ARNT point mutation (Gly326→Asp) in Hepa1C4 cellshas recently been reported, which leads to increasedproteolytic susceptibility and a higher turnover rate(Numayama-Tsuruta et al., 1997). Despite reduced ARNTmRNA and protein levels, as well as lack of HIF-1 DNA-binding and HIF-1-mediated gene activation (Gassmann etal., 1997; Gradin et al., 1996; Wenger et al., 1998), thisARNT mutation did not abrogate its ability to translocate tothe nucleus and to form heterodimers with AhR (Numayama-Tsuruta et al., 1997). Thus, residual amounts of ARNT mighthave been responsible for the nuclear localization of HIF-1αobserved in Hepa1C4 cells (Fig. 5). To rule out thispossibility, ES cells in which the ARNT gene has beentargeted by homologous recombination (Maltepe et al., 1997)were examined. As shown in Fig. 7A, HIF-1α was detectedby immunoblotting in nuclear extracts of hypoxic ARNT+/+as well as ARNT−/− ES cells, albeit at a lower level in thelatter cell line. Nuclear ARNT levels were increased inhypoxic ARNT+/+ ES cells, but, as expected, could not be

detected in ARNT−/− ES cells. Staining of ARNT+/+ andARNT−/− ES cells with the anti-HIF-1α IgY antibody andanalysis by CLSM revealed a weak homogenousimmunofluorescence in normoxic cells (Fig. 7Bb,f). Inhypoxic wild-type ES cells, HIF-1α accumulated in the non-nucleolar part of the nuclei (Fig. 7Bc,d). Nuclearaccumulation of HIF-1α was also clearly observed in hypoxicARNT−/− ES cells. As can be seen in Fig. 7Bg, HIF-1αsignals were variable from cell to cell. However, usingCLSM, scanning through multiple horizontal sections of thecell aggregates revealed that the HIF-1α was present in thenuclei of a majority of the cells (data not shown). Thus, thesedata confirmed that whereas hypoxic HIF-1α accumulationand nuclear translocation is ARNT-independent, HIF-1αstability in the nucleus during isolation of the nuclei islowered in the absence of ARNT.

Subcellular localization of HIF-1 α followingproteasome inhibition and thiol reductionSalceda and Caro (1997) reported that the proteasome inhibitorlactacystin and the thiol-reducing agent N-(2-mercaptopropionyl)-glycine (NMPG) induced HIF-1α proteinin normoxic cells. It is conceivable that additional hypoxia-inducible post-stabilization steps (e.g. phosphorylation) might

Fig. 7.Hypoxic HIF-1α nuclear accumulation in ARNT+/+ and ARNT−/− embryonic stem (ES) cells. (A) Nuclear extracts (n) and cytoplasmicfractions (c) from normoxic and hypoxic ARNT+/+ and ARNT−/− ES cells were analyzed by immunoblotting using the anti-HIF-1α IgY or therabbit anti-ARNT antibody. (B) Indirect immunofluorescence analysis of normoxic and hypoxic ARNT+/+ (b,c) and ARNT−/− (f,g) ES cellsusing the anti-HIF-1α IgY antibody. The corresponding phase-contrast images are shown in the outer columns.

Page 8: Subcellular localization of HIF-1(alpha) and ARNTjcs.biologists.org/content/joces/112/8/1203.full.pdf · HIF-1βis identical to the aryl hydrocarbon receptor nuclear translocator

1210

be required for nuclear translocation. Therefore, we firstassessed HIF-1α protein induction in total cellular lysatesderived from HeLa cells. As shown in Fig. 8A, lactacystin andNMPG induced a series of HIF-1α isoforms with differentmobilities compared to hypoxically induced HIF-1α,indicating alternative post-translational modifications.However, nuclear accumulation of HIF-1α was observed byimmunofluorescence as a result of the treatment with all ofthese stimuli (Fig. 8B). In addition, we found that the ironchelator desferrioxamine induced protein stability and nuclearaccumulation of HIF-1α (data not shown). Thus, hypoxia aswell as hypoxia-mimicking agents that interfere with theprocess of protein modification/degradation lead to an increasein protein levels that is sufficient for nuclear translocation ofHIF-1α.

DISCUSSION

In the present study, we used ARNT-mutant Hepa1C4 cells(Numayama-Tsuruta et al., 1997) as well as ARNT-deficientES cells (Maltepe et al., 1997) to demonstrate that HIF-1αprotein accumulation and nuclear translocation can occurindependently of ARNT. This finding is not compatible withthe term ‘nuclear translocator’ in ‘ARNT’. There is a similardiscrepancy between biochemical fractionation andimmunofluorescence data, as we show in this work for the HIF-1α:ARNT complex, which has also been reported for theAhR:ARNT complex (reviewed in Hankinson, 1995; Schmidtand Bradfield, 1996). In Hepa1, but not in Hepa1C4 cells, theliganded AhR can be purified from the nucleus. Thecomplementing ARNT was hence believed to be responsiblefor nuclear translocation of AhR. That this model is wrong hasbeen demonstrated by immunofluorescence experiments

showing that AhR and ARNT are translocated to the nucleusindependently (Hord and Perdew, 1994; Pollenz et al., 1994),and that following ligand binding the AhR translocates to thenucleus where it is rapidly depleted, whereas ARNT remainsat constant levels in the nucleus (Pollenz, 1996). Thus, uponhomogenization almost all of the ARNT present in a cell isfound outside of the nuclei, apparently because it leaches outof the nuclei (Hord and Perdew, 1994).

A plausible explanation for the failure of subcellularfractionation methods to accurately preserve the distribution ofAhR and ARNT observed in vivo might be that heterodimerformation in the nucleus renders the complex more stablyassociated with the nuclear compartment during isolation of thenuclei. Hence monomeric subunits are more prone to leakage.In this work, we found partial loss of both HIF-1 subunitsduring isolation of the nuclei in the absence of one of therespective heterodimerization partners in Hepa1C4 and EScells (lacking ARNT) or under normoxic conditions (lackingHIF-1α). Whether this is due to interaction of the heterodimerwith the nuclear scaffold and/or specific DNA binding is amatter of further investigations. In this context, it is noteworthythat Poellinger and coworkers reported a conformationalchange of HIF-1α upon interaction with ARNT, as shown byaltered resistance to proteolytic digestion in vitro (Kallio et al.,1997). Even if this effect might be due to steric hindrance, itprovides evidence that increased proteolytic stability in vivoduring isolation of the nuclei could also contribute to thedifference observed in monomeric versus heterodimericprotein levels of the HIF-1 subunits.

Our findings on ARNT-independent HIF-1α induction inHepa1C4 and ARNT−/− ES cells are in striking contrast to arecent publication where the same cell lines were used as HIF-1α-deficient cell culture models (An et al., 1998). Therein, theauthors claimed that HIF-1α hypoxically stabilizes wild-type

D. Chilov and others

Fig. 8.Hypoxic nuclear accumulation of HIF-1α canbe mimicked by lactacystin and NMPG. (A) HeLacells were exposed for 6 hours to either 1% O2, 10,20 or 80 µM lactacystin, or to 1 mM NMPG, andanalyzed by immunoblotting using the anti-HIF-1αIgY antibody. (B) Indirect immunofluorescenceanalysis of HeLa cells exposed for 6 hours to either1% O2 (hypoxia), 20 µM lactacystin or 1 mMNMPG. Despite the stronger induction of lactacystinat 80 µM (Fig. 8A), this concentration was not usedbecause it was toxic and interfered with the structuralintegrity of the cells (not shown). The cells wereprepared and stained with the anti-HIF-1α IgYantibody as described in Materials and methods.

Page 9: Subcellular localization of HIF-1(alpha) and ARNTjcs.biologists.org/content/joces/112/8/1203.full.pdf · HIF-1βis identical to the aryl hydrocarbon receptor nuclear translocator

1211Subcellular localization of HIF-1α and ARNT

p53 protein, as substantiated by the lack of hypoxic p53induction in the ARNT-deficient cell lines. However, sincehypoxia normally induces HIF-1α in these cells, the simplifiedmodel of increased p53 due to interaction with HIF-1α needsrefinement (Wenger et al., 1998).

Recently, nuclear localization signals (NLS) were identifiedin the amino termini of AhR, ARNT and HIF-1α as well as asecond NLS in the carboxy-terminal part of HIF-1α (Eguchiet al., 1997; Ikuta et al., 1998; Kallio et al., 1998), consistentwith their ability to translocate to the nucleus independently.However, these data were based on transient transfectionexperiments with fusion protein vectors, conditions that leadto unphysiological accumulation of products already innormoxia, probably because of the high copy number oftransfected plasmids or because the fusion partners maystabilize the HIF-1α protein (Kallio et al., 1998). In contrast,we showed that endogenous HIF-1α can accumulate andtranslocate to the nucleus in hypoxic conditions only.Interestingly, a nuclear export signal was also identified in theconserved bHLH region of AhR (Ikuta et al., 1998). Given therapid degradation of HIF-1α upon reoxygenation (Huang et al.,1996), it will be important to determine whether HIF-1αdegradation occurs inside or outside the nucleus, probablyinvolving a nuclear export signal.

We are grateful to E. G. Van Meir, O. Hankinson, V. O’Donnall, L.Poellinger, J. Caro, C. Simon and G. H. Perdew for the generous giftof cell lines, plasmids and antibodies; to P. Spielmann and F. Parpanfor excellent technical assistance; to S. Kunz for advice; to D. Strokaand I. Desbaillets for critically reading the manuscript; to C. Gasserfor the artwork; and to C. Bauer for support. This work was supportedby the Swiss National Science Foundation (31-47111.96) and theWolfermann-Nägeli-Stiftung. R.H.W. is a recipient of the‘Sondermassnahmen des Bundes zur Förderung des akademischenNachwuchses’.

REFERENCES

An, W. G., Kanekal, M., Simon, M. C., Maltepe, E., Blagosklonny, M. V.and Neckers, L. M. (1998). Stabilization of wild-type p53 by hypoxia-inducible factor 1α. Nature392, 405-408.

Bunn, H. F. and Poyton, R. O.(1996). Oxygen sensing and molecularadaptation to hypoxia. Physiol. Rev.76, 839-885.

Camenisch, G., Tini, M., Chilov, D., Kvietikova, I., Srinivas, V., Caro, J.,Spielmann, P., Wenger, R. H. and Gassmann, M.(1999). Generalapplicability of chicken egg yolk antibodies: the performance of IgYimmunoglobulins raised against the hypoxia-inducible factor 1α. FASEB J.13, 81-88.

Carmeliet, P., Dor, Y., Herbert, J. M., Fukumura, D., Brusselmans, K.,Dewerchin, M., Neeman, M., Bono, F., Abramovitch, R., Maxwell, P. etal. (1998). Role of HIF-1α in hypoxia-mediated apoptosis, cell proliferationand tumour angiogenesis. Nature394, 485-490.

Eguchi, H., Ikuta, T., Tachibana, T., Yoneda, Y. and Kawajiri, K. (1997).A nuclear localization signal of human aryl hydrocarbon receptor nucleartranslocator/hypoxia-inducible factor 1β is a novel bipartite type recognizedby the two components of nuclear pore-targeting complex. J. Biol. Chem.272, 17640-17647.

Forsythe, J. A., Jiang, B. H., Iyer, N. V., Agani, F., Leung, S. W., Koos, R.D. and Semenza, G. L.(1996). Activation of vascular endothelial growthfactor gene transcription by hypoxia-inducible factor 1. Mol. Cell. Biol.16,4604-4613.

Gassmann, M., Kvietikova, I., Rolfs, A. and Wenger, R. H.(1997). Oxygen-and dioxin-regulated gene expression in mouse hepatoma cells. Kidney Int.51, 567-574.

Gassmann, M. and Wenger, R. H.(1997). HIF-1, a mediator of the molecularresponse to hypoxia. News Physiol. Sci.12, 214-218.

Gradin, K., McGuire, J., Wenger, R. H., Kvietikova, I., Whitelaw, M. L.,Toftgård, R., Tora, L., Gassmann, M. and Poellinger, L. (1996).Functional interference between hypoxia and dioxin signal transductionpathways: competition for recruitment of the Arnt transcription factor. Mol.Cell. Biol. 16, 5221-5231.

Hankinson, O. (1995). The aryl hydrocarbon receptor complex. Annu. Rev.Pharmacol. Toxicol.35, 307-340.

Hoffman, E. C., Reyes, H., Chu, F. F., Sander, F., Conley, L. H., Brooks,B. A. and Hankinson, O.(1991). Cloning of a factor required for activityof the Ah (dioxin) receptor. Science252, 954-958.

Hord, N. G. and Perdew, G. H. (1994). Physicochemical andimmunocytochemical analysis of the aryl hydrocarbon receptor nucleartranslocator: characterization of two monoclonal antibodies to the arylhydrocarbon receptor nuclear translocator. Mol. Pharmacol.46, 618-626.

Huang, E. L., Gu, J., Schau, M. and Bunn, H. F.(1998). Regulation ofhypoxia-inducible factor 1α is mediated by an oxygen-dependent domainvia the ubiquitin-proteasome pathway. Proc. Natl. Acad. Sci. USA95, 7987-7992.

Huang, L. E., Arany, Z., Livingston, D. M. and Bunn, H. F. (1996).Activation of hypoxia-inducible transcription factor depends primarily uponredox-sensitive stabilization of its α subunit. J. Biol. Chem.271, 32253-32259.

Ikuta, T., Eguchi, H., Tachibana, T., Yoneda, Y. and Kawajiri, K. (1998).Nuclear localization and export signals of the human aryl hydrocarbonreceptor. J. Biol. Chem.273, 2895-2904.

Iyer, N. V., Kotch, L. E., Agani, F., Leung, S. W., Laughner, E., Wenger,R. H., Gassmann, M., Gearhart, J. D., Lawler, A. M., Yu, A. Y. et al.(1998). Cellular and developmental control of O2 homeostasis by hypoxia-inducible factor 1α. Genes Dev.12, 149-162.

Jiang, B. H., Agani, F., Passaniti, A. and Semenza, G. L.(1997a). V-SRCinduces expression of hypoxia-inducible factor 1 (HIF-1) and transcriptionof genes encoding vascular endothelial growth factor and enolase 1:involvement of HIF-1 in tumor progression. Cancer Res.57, 5328-5335.

Jiang, B. H., Rue, E., Wang, G. L., Roe, R. and Semenza, G. L.(1996a).Dimerization, DNA binding, and transactivation properties of hypoxia-inducible factor 1. J. Biol. Chem.271, 17771-17778.

Jiang, B. H., Semenza, G. L., Bauer, C. and Marti, H. H.(1996b). Hypoxia-inducible factor 1 levels vary exponentially over a physiologically relevantrange of O2 tension. Am. J. Physiol.271, C1172-1180.

Jiang, B. H., Zheng, J. Z., Leung, S. W., Roe, R. and Semenza, G. L.(1997b). Transactivation and inhibitory domains of hypoxia-inducible factor1α. Modulation of transcriptional activity by oxygen tension. J. Biol. Chem.272, 19253-19260.

Kallio, P. J., Okamoto, K., O’Brien, S., Carrero, P., Makino, Y., Tanaka,H. and Poellinger, L.(1998). Signal transduction in hypoxic cells: induciblenuclear translocation and recruitment of the CBP/p300 coactivator by thehypoxia-inducible factor-1α. EMBO J.17, 6573-6586.

Kallio, P. J., Pongratz, I., Gradin, K., McGuire, J. and Poellinger, L.(1997). Activation of hypoxia-inducible factor 1α: posttranscriptionalregulation and conformational change by recruitment of the Arnttranscription factor. Proc. Natl. Acad. Sci. USA94, 5667-5672.

Kozak, K. R., Abbott, B. and Hankinson, O.(1997). Arnt-deficient mice andplacental differentiation. Dev. Biol.191, 297-305.

Kvietikova, I., Wenger, R. H., Marti, H. H. and Gassmann, M.(1995). Thetranscription factors ATF-1 and CREB-1 bind constitutively to the hypoxia-inducible factor-1 (HIF-1) DNA recognition site. Nucl. Acids Res.23, 4542-4550.

Lee, P. J., Jiang, B. H., Chin, B. Y., Iyer, N. V., Alam, J., Semenza, G. L.and Choi, A. M. (1997). Hypoxia-inducible factor-1 mediatestranscriptional activation of the heme oxygenase-1 gene in response tohypoxia. J. Biol. Chem.272, 5375-5381.

Liu, Y. X., Christou, H., Morita, T., Laughner, E., Semenza, G. L. andKourembanas, S.(1998). Carbon monoxide and nitric oxide suppress thehypoxic induction of vascular endothelial growth factor gene via the 5′enhancer. J. Biol. Chem.273, 15257-15262.

Maltepe, E., Schmidt, J. V., Baunoch, D., Bradfield, C. A. and Simon, M.C. (1997). Abnormal angiogenesis and responses to glucose and oxygendeprivation in mice lacking the protein ARNT. Nature386, 403-407.

Martin, C., Yu, A. Y., Jiang, B. H., Davis, L., Kimberly, D., Hohimer, A.R. and Semenza, G. L.(1998). Cardiac hypertrophy in chronically anemicfetal sheep – increased vascularization is associated with increasedmyocardial expression of vascular endothelial growth factor and hypoxia-inducible factor 1. Am. J. Obst. Gynecol.178, 527-534.

Numayama-Tsuruta, K., Kobayashi, A., Sogawa, K. and Fujii-Kuriyama,

Page 10: Subcellular localization of HIF-1(alpha) and ARNTjcs.biologists.org/content/joces/112/8/1203.full.pdf · HIF-1βis identical to the aryl hydrocarbon receptor nuclear translocator

1212

Y. (1997). A point mutation responsible for defective function of the aryl-hydrocarbon-receptor nuclear translocator in mutant Hepa-1c1c7 cells. Eur.J. Biochem.246, 486-495.

Pollenz, R. S. (1996). The aryl-hydrocarbon receptor, but not the aryl-hydrocarbon receptor nuclear translocator protein, is rapidly depleted inhepatic and nonhepatic culture cells exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Mol. Pharmacol.49, 391-398.

Pollenz, R. S., Sattler, C. A. and Poland, A.(1994). The aryl hydrocarbonreceptor and aryl hydrocarbon receptor nuclear translocator protein showdistinct subcellular localizations in Hepa 1c1c7 cells byimmunofluorescence microscopy. Mol. Pharmacol.45, 428-438.

Ryan, H. E., Lo, J. and Johnson, R. S.(1998). HIF-1α is required forsolid tumor formation and embryonic vascularization. EMBO J.17, 3005-3015.

Salceda, S., Beck, I. and Caro, J.(1996). Absolute requirement of arylhydrocarbon receptor nuclear translocator protein for gene activation byhypoxia. Arch. Biochem. Biophys.334, 389-394.

Salceda, S. and Caro, J.(1997). Hypoxia-inducible factor 1α (HIF-1α)protein is rapidly degraded by the ubiquitin-proteasome system undernormoxic conditions. Its stabilization by hypoxia depends on redox-inducedchanges. J. Biol. Chem.272, 22642-22647.

Schmidt, J. V. and Bradfield, C. A.(1996). Ah receptor signaling pathways.Ann. Rev. Cell. Dev. Biol.12, 55-89.

Semenza, G. L.(1998). Hypoxia-inducible factor 1: master regulator of O2homeostasis. Curr. Opin. Genet. Dev.8, 588-594.

Tian, H., Hammer, R. E., Matsumoto, A. M., Russell, D. W. and McKnight,S. L. (1998). The hypoxia-responsive transcription factor EPAS1 is essentialfor catecholamine homeostasis and protection against heart failure duringembryonic development. Genes Dev.12, 3320-3324.

Wang, G. L., Jiang, B. H., Rue, E. A. and Semenza, G. L.(1995a). Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated bycellular O2 tension. Proc. Natl. Acad. Sci. USA92, 5510-5514.

Wang, G. L., Jiang, B. H. and Semenza, G. L.(1995b). Effect of alteredredox states on expression and DNA-binding activity of hypoxia-induciblefactor 1. Biochem. Biophys. Res. Commun.212, 550-556.

Wang, G. L., Jiang, B. H. and Semenza, G. L.(1995c). Effect of proteinkinase and phosphatase inhibitors on expression of hypoxia-inducible factor1. Biochem. Biophys. Res. Commun.216, 669-675.

Wenger, R. H., Camenisch, G., Desbaillets, I., Chilov, D. and Gassmann,M. (1998). Up-regulation of hypoxia-inducible factor-1α is not sufficientfor hypoxic/anoxic p53 induction. Cancer Res.58, 5678-5680.

Wenger, R. H. and Gassmann, M.(1997). Oxygen(es) and the hypoxia-inducible factor-1. Biol. Chem.378, 609-616.

Wenger, R. H., Kvietikova, I., Rolfs, A., Gassmann, M. and Marti, H. H.(1997). Hypoxia-inducible factor-1α is regulated at the post-mRNA level.Kidney Int.51, 560-563.

Wood, S. M., Gleadle, J. M., Pugh, C. W., Hankinson, O. and Ratcliffe, P.J. (1996). The role of the aryl hydrocarbon receptor nuclear translocator(ARNT) in hypoxic induction of gene expression. Studies in ARNT-deficient cells. J. Biol. Chem.271, 15117-15123.

D. Chilov and others