Hypoxia-inducible Factor 1α (HIF-1α) Protein Is Rapidly Degraded by the Ubiquitin-Proteasome System under Normoxic Conditions

Abstract

The hypoxia-inducible factor 1 transcriptional activator complex (HIF-1) is involved in the activation of the erythropoietin and several other hypoxia-responsive genes. The HIF-1 complex is composed of two protein subunits: HIF-1β/ARNT (aryl hydrocarbon receptor nuclear translocator), which is constitutively expressed, and HIF-1α, which is not present in normal cells but induced under hypoxic conditions. The HIF-1α subunit is continuously synthesized and degraded under normoxic conditions, while it accumulates rapidly following exposure to low oxygen tensions. The involvement of the ubiquitin-proteasome system in the proteolytic destruction of HIF-1 in normoxia was studied by the use of specific inhibitors of the proteasome system. Lactacystin and MG-132 were found to protect the degradation of the HIF-1 complex in cells transferred from hypoxia to normoxia. The same inhibitors were able to induce HIF-1 complex formation when added to normoxic cells. Final confirmation of the involvement of the ubiquitin-proteasome system in the regulated degradation of HIF-1α was obtained by the use ofts20TG R cells, which contain a temperature-sensitive mutant of E1, the ubiquitin-activating enzyme. Exposure of ts20 cells, under normoxic conditions, to the non-permissive temperature induced a rapid and progressive accumulation of HIF-1. The effect of proteasome inhibitors on the normoxic induction of HIF-1 binding activity was mimicked by the thiol reducing agentN-(2-mercaptopropionyl)-glycine and by the oxygen radical scavenger 2-acetamidoacrylic acid. Furthermore,N-(2-mercaptopropionyl)-glycine induced gene expression as measured by the stimulation of a HIF-1-luciferase expression vector and by the induction of erythropoietin mRNA in normoxic Hep 3B cells. These last findings strongly suggest that the hypoxia induced changes in HIF-1α stability and subsequent gene activation are mediated by redox-induced changes. The hypoxia-inducible factor 1 transcriptional activator complex (HIF-1) is involved in the activation of the erythropoietin and several other hypoxia-responsive genes. The HIF-1 complex is composed of two protein subunits: HIF-1β/ARNT (aryl hydrocarbon receptor nuclear translocator), which is constitutively expressed, and HIF-1α, which is not present in normal cells but induced under hypoxic conditions. The HIF-1α subunit is continuously synthesized and degraded under normoxic conditions, while it accumulates rapidly following exposure to low oxygen tensions. The involvement of the ubiquitin-proteasome system in the proteolytic destruction of HIF-1 in normoxia was studied by the use of specific inhibitors of the proteasome system. Lactacystin and MG-132 were found to protect the degradation of the HIF-1 complex in cells transferred from hypoxia to normoxia. The same inhibitors were able to induce HIF-1 complex formation when added to normoxic cells. Final confirmation of the involvement of the ubiquitin-proteasome system in the regulated degradation of HIF-1α was obtained by the use ofts20TG R cells, which contain a temperature-sensitive mutant of E1, the ubiquitin-activating enzyme. Exposure of ts20 cells, under normoxic conditions, to the non-permissive temperature induced a rapid and progressive accumulation of HIF-1. The effect of proteasome inhibitors on the normoxic induction of HIF-1 binding activity was mimicked by the thiol reducing agentN-(2-mercaptopropionyl)-glycine and by the oxygen radical scavenger 2-acetamidoacrylic acid. Furthermore,N-(2-mercaptopropionyl)-glycine induced gene expression as measured by the stimulation of a HIF-1-luciferase expression vector and by the induction of erythropoietin mRNA in normoxic Hep 3B cells. These last findings strongly suggest that the hypoxia induced changes in HIF-1α stability and subsequent gene activation are mediated by redox-induced changes. Mammalian cells are able to sense oxygen tension and turn on a series of genes in response to the lack of oxygen. The best characterized of these hypoxia-regulated genes is the one coding for erythropoietin (Epo), 1The abbreviations used are: Epo, erythropoietin; HIF-1, hypoxia-inducible factor 1; ARNT, aryl hydrocarbon receptor nuclear translocator; NMPG, N-(2-mercaptopropionyl)-glycine; PAS, PER-ARNT-SIM; Ac, acetyl; Z, benzyloxycarbonyl; E1, Ub-activating enzyme.1The abbreviations used are: Epo, erythropoietin; HIF-1, hypoxia-inducible factor 1; ARNT, aryl hydrocarbon receptor nuclear translocator; NMPG, N-(2-mercaptopropionyl)-glycine; PAS, PER-ARNT-SIM; Ac, acetyl; Z, benzyloxycarbonyl; E1, Ub-activating enzyme. the growth factor that regulates red cell production (reviewed in Ref. 1Ratcliffe P.J. Ebert B.L. Firth J.D. Gleadle J.M. Maxwell P.H. Nagao M. O'Rourke J.F. Pugh C.W. Wood S.M. Kidney Int. 1997; 51: 514-526Abstract Full Text PDF PubMed Scopus (45) Google Scholar). The hypoxia response of the Epo gene is controlled by an enhancer element located in the 3′-flanking region of the gene (2Beck I. Ramirez S. Weinmann R. Caro J. J. Biol. Chem. 1991; 266: 15563-15566Abstract Full Text PDF PubMed Google Scholar, 3Semenza G.L. Nejfelt M.K. Chi S.M. Antonarakis S.E. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5680-5684Crossref PubMed Scopus (704) Google Scholar, 4Pugh C.W. Tan C.C. Jones R.W. Ratcliffe P.J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10553-10557Crossref PubMed Scopus (225) Google Scholar). Transcriptional activation of the enhancer is mediated by a hypoxia-inducible DNA-binding protein complex termed HIF-1, which binds to the site-1 sequences of the enhancer (5Semenza G.L. Wang G.L. Mol. Cell. Biol. 1992; 12: 5447-5454Crossref PubMed Scopus (2174) Google Scholar, 6Beck I. Weinmann R. Caro J. Blood. 1993; 82: 704-711Crossref PubMed Google Scholar). Similar enhancer elements, also involving the binding of HIF-1, have been identified in other hypoxia-responsive genes, such as those coding for vascular endothelial growth factor (7Levy A.P. Levy N.S. Wegner S. Goldberg M.A. J. Biol. Chem. 1995; 270: 13333-13340Abstract Full Text Full Text PDF PubMed Scopus (877) Google Scholar), glucose transporter-1, and several glycolytic enzymes (8Firth J.D. Ebert B.L. Pugh C.W. Ratcliffe P.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6496-6500Crossref PubMed Scopus (443) Google Scholar, 9Semenza G.L. Roth P.H. Fang H.-M. Wang G.L. J. Biol. Chem. 1994; 269: 23757-23763Abstract Full Text PDF PubMed Google Scholar, 10Ebert B.L. Firth J.D. Ratcliffe P.J. J. Biol. Chem. 1995; 270: 29083-29089Abstract Full Text Full Text PDF PubMed Scopus (440) Google Scholar). All these genes also respond like Epo, to cobalt ions and iron chelators, suggesting a common mechanism for oxygen sensing and gene activation. The recent cloning of the protein components of the HIF-1 complex identified two subunits, HIF-1α and HIF-1β, which belong to the subfamily of basic helix-loop-helix transcription factors containing a PAS (PER-ARNT-SIM) motif (11Wang G.L. Jiang B.-H. Rue E.A. Semenza G.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5510-5514Crossref PubMed Scopus (4995) Google Scholar). The HIF-1α subunit is a new member of the family, whereas HIF-1β corresponds to the known aryl hydrocarbon receptor nuclear translocator (ARNT) protein. Hypoxia induces the formation of HIF-1 complex by a process that requires protein synthesis (5Semenza G.L. Wang G.L. Mol. Cell. Biol. 1992; 12: 5447-5454Crossref PubMed Scopus (2174) Google Scholar). The mechanisms by which cells sense the lack of oxygen and initiate the hypoxic response are currently unknown. However, significant indirect evidence suggests that redox-mediated processes are likely involved in this step. Treatment of cells with hydrogen peroxide greatly reduces HIF-1 formation and Epo mRNA expression in response to hypoxic stimulation (12Fandrey J. Frede S. Jelkmann W. Biochem. J. 1994; 303: 507-510Crossref PubMed Scopus (224) Google Scholar, 13Wang G.L. Jiang B.-H. Semenza G.L. Biochem. Biophys. Res. Commun. 1995; 212: 550-556Crossref PubMed Scopus (158) Google Scholar). Since oxygen radicals and superoxide formation are very dependent on oxygen availability, their reduced formation under hypoxic conditions could serve as the initial signal in oxygen sensing. Of the components of the HIF-1 complex, ARNT protein is constitutively expressed in all cells while HIF-1α is present only in hypoxic cells. Thus, HIF-1 complex formation appears to be determined primarily by the abundance of the HIF-1α subunit. The observation that hypoxia does not modify HIF-1α mRNA levels suggested that HIF-1α protein content is regulated at the level of its mRNA translation or by changes in its rate of degradation (14Salceda S. Beck I. Caro J. Arch. Biochem. Biophys. 1996; 334: 389-394Crossref PubMed Scopus (83) Google Scholar). Indeed, Huang et al. (15Huang L.E. Arany Z. Livingston D.M. Bunn H.F. J. Biol. Chem. 1996; 271: 32253-32259Abstract Full Text Full Text PDF PubMed Scopus (1018) Google Scholar) recently reported that HIF-1α protein is highly unstable under normoxic conditions and that hypoxia significantly prolonged its half-life, thus allowing its accumulation and the formation of the complex. The mechanisms involved in the rapid degradation of HIF-1α under normoxic conditions and the signals involved in the stabilization process by hypoxia, are currently unknown. The results presented here indicate that the rapid degradation of HIF-1α under normoxic conditions is mediated by the ubiquitin-proteasome system and its stabilization is probably induced by redox-mediated changes. Hep 3B and B-1 cells were cultured in minimal essential medium (Life Technologies, Inc., Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum (Hyclone, Logan, UT), penicillin (100 units/ml), and streptomycin (100 μg/ml) (Life Technologies, Inc.). Cells were maintained at 37 °C in an atmosphere of 5% CO2. Hep 3B cells were obtained from the American Tissue Culture Collection. The B-1 cell line is a Hep 3B-derived cell line which was stably transfected with an expression vector containing luciferase cDNA under the control of a minimal Epo promoter (330-base pair SfaNI-XbaIII fragment) and the hypoxia responsive enhancer from the human Epo gene (150-base pair ApaI/PstI fragment). The response of these cells to hypoxia, cobalt, and desferrioxamine has been reported (16Salceda S. Beck I. Srinivas V. Caro J. Kidney Int. 1997; 51: 556-559Abstract Full Text PDF PubMed Scopus (59) Google Scholar). For hypoxic stimulation cells were flushed with a gas mixture containing 0.5% O2, 5% CO2 and balanced N2 as already described (2Beck I. Ramirez S. Weinmann R. Caro J. J. Biol. Chem. 1991; 266: 15563-15566Abstract Full Text PDF PubMed Google Scholar). The BALB/c 3T3 andts20TG R (17Chowdary D.R. Dermody J.J. Jha K.K. Ozer H.L. Mol. Cell. Biol. 1994; 14: 1997-2003Crossref PubMed Scopus (266) Google Scholar) cell lines were provided by Dr. Harvey L. Ozer, Department of Microbiology and Molecular Genetics, UMDNJ, New Jersey Medical School, Newark, New Jersey. Both cell lines were maintained at 35 °C in a humidified incubator with 10% CO2 in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum and antibiotics as described above. The permissive and non-permissive temperatures for the ts20TG R mutant cell line are 35 and 39 °C, respectively. For inhibitor experiments, calpain inhibitors I and II (Calbiochem-Novabiochem Corp., La Jolla, CA) were dissolved in ethanol. MG-132 (Peptides International, Inc., Knoxville, KY), E-64d (Sigma), interleukin-1β-converting enzyme inhibitor II (BACHEM Bioscience, Inc., King of Prussia, PA), and lactacystin (provided by Dr. S. Ōmura from The Kitasato Institute, Tokyo, Japan) (18Ōmura S. Fujimoto T. Otoguro K. Matsuzaki K. Moriguchi R. Tanaka H. Sasaki Y. J. Antibiot. ( Tokyo ). 1991; 44: 113-116Crossref PubMed Scopus (532) Google Scholar) were dissolved in dimethyl sulfoxide. Control cells were untreated or treated with dimethyl sulfoxide or ethanol. No differences in binding activity were found among these samples. Catalase, AD-1, and NMPG were from Sigma. Nuclear extracts were prepared from normal or treated cells as described previously (14Salceda S. Beck I. Caro J. Arch. Biochem. Biophys. 1996; 334: 389-394Crossref PubMed Scopus (83) Google Scholar). Electrophoretic mobility shift assay was performed by incubating 7 μg of nuclear extract with32P-labeled double-stranded oligonucleotide probe as described previously (6Beck I. Weinmann R. Caro J. Blood. 1993; 82: 704-711Crossref PubMed Google Scholar). For supershift assays, 1 μl of polyclonal antiserum raised in rabbits against recombinant HIF-1α or ARNT (1:5 dilution) were added to the nuclear extracts and incubated for 2 h on ice prior to adding labeled probe. Antibodies were kindly provided by Drs. G. L. Semenza (The Johns Hopkins University, School of Medicine, Baltimore, Maryland) and C. A. Bradfield (Department of Oncology, Medical School, University of Wisconsin-Madison, Madison, Wisconsin). A normal rabbit serum (preimmune) served as a negative antibody control. All cell extracts were prepared and analyzed using the Luciferase Assay System (Promega, Madison, WI). Briefly, 35-mm plates were washed twice with cold 1 × phosphate-buffered saline and 100 μl of 1 × lysis buffer was then added to the cells. Samples were collected and 5-μl aliquots were assayed using luciferase assay reagent. Luminescence was measured in a TD 20/20 luminometer (Promega), and results expressed as relative light units per μg of total protein. Protein concentrations were determined by a commercial kit (Bio-Rad), using bovine serum albumin as the standard. Total RNA was extracted by utilizing the as described by and Biochem. PubMed Scopus Google Scholar). For μg of total RNA was in a transferred to and by were in buffer at was performed in the same containing 1 × of probe at the same were washed twice with 2 × at temperature and with × at °C by exposure to probe was obtained from a human cDNA as already described C.W. Tan C.C. Jones R.W. Ratcliffe P.J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10553-10557Crossref PubMed Scopus (225) Google Scholar) and labeled with using a translation kit (Life Technologies, Inc.). by Wang et al. (11Wang G.L. Jiang B.-H. Rue E.A. Semenza G.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5510-5514Crossref PubMed Scopus (4995) Google Scholar) that following of hypoxia is a rapid of HIF-1α protein and HIF-1 complex with a of the of proteolytic degradation in this rapid a series of inhibitors with enzyme Nuclear extracts were obtained from Hep 3B cells with hypoxia for h and then transferred to normoxia for an inhibitors were added the last of the hypoxic for lactacystin that it was added 1 h the and while in normoxia. in the shift in from hypoxia to normoxia a rapid of the HIF-1 complex which was by the of the in normoxia. of the inhibitor to a inhibitor this whereas a and a highly specific The from hypoxia to normoxia and the of inhibitors effect on the expression of binding activity 1 The effect of calpain inhibitor which also has activity against the and the lack of effect of E-64d suggested that the ubiquitin-proteasome system was likely involved in HIF-1 The of the proteasome was using the inhibitors and the as in in this is the lack of effect of the an inhibitor of enzyme of proteasome inhibitors on HIF-1 binding of as in shift using HIF-1 probe. of hypoxic of normoxia are: and shift in utilizing The results indicate that the rapid of HIF-1 complex of hypoxia could be by the use of these inhibitors could also induce the formation of HIF-1 complex in normoxic cells. For this Hep 3B cells were incubated under normoxic conditions for h in the of inhibitors and their nuclear extracts assayed for HIF-1 activity by shift in calpain inhibitor and to a calpain inhibitor and the formation of HIF-1 complex in normoxic cells, whereas and E-64d effect these effect on of the of HIF-1α and ARNT in the induced was obtained by supershift utilizing specific against protein subunits, as in C. using that lactacystin the level of in transfected normoxic cells. Srinivas and J. The of changes in HIF-1 induction was studied in normoxic Hep 3B cells to the agentN-(2-mercaptopropionyl)-glycine in of cells with NMPG for h induced HIF-1 complex, while effect was in A effect of NMPG on the proteasome system was by the that NMPG not the expression of whereas the proteasome inhibitors induced it in the of HIF-1α and ARNT in the complex. the effect of NMPG on gene activation a Hep 3B-derived cell line stably transfected with a luciferase expression vector containing a minimal Epo promoter and a These cells have been to respond to hypoxia by luciferase expression in a (16Salceda S. Beck I. Srinivas V. Caro J. Kidney Int. 1997; 51: 556-559Abstract Full Text PDF PubMed Scopus (59) Google Scholar). Exposure of B-1 cells to concentrations of NMPG for h a stimulation of luciferase as in A. Similar results were found when the oxygen radical scavenger 2-acetamidoacrylic was used A effect NMPG and hypoxia is in B-1 cells were to NMPG at or 0.5% for The effect of NMPG was also mimicked by the of which and its confirmation of the effect of NMPG on gene expression was obtained by of RNA obtained from normoxic Hep 3B cells treated with NMPG for as in the proteasome inhibitors not Epo No changes in HIF-1α mRNA levels were found in untreated or hypoxia, proteasome and Hep 3B cells of is the in the degradation of by the proteasome system. a basic protein of found in all cells, be to in an Ub-activating enzyme this in the formation of an the involvement of the ubiquitin-proteasome in the proteolytic degradation of HIF-1α under normoxic conditions a BALB/c cell containing a temperature-sensitive mutant of (17Chowdary D.R. Dermody J.J. Jha K.K. Ozer H.L. Mol. Cell. Biol. 1994; 14: 1997-2003Crossref PubMed Scopus (266) Google Scholar). Cells cultured under the permissive temperature a whereas the shift to the non-permissive temperature For these experiments, ts20 cells were cultured under normoxic conditions at 35 and 39 °C for and h and nuclear extracts were obtained and for HIF-1 complex formation by shift in cells at 35 °C not HIF-1, whereas the shift to the non-permissive temperature a progressive accumulation of the complex. No were on binding against HIF-1α and ARNT the of the complex as HIF-1. Similar with the 3T3 cell line induction of HIF-1 with the temperature Hypoxia of the Epo and other genes are mediated by the binding of a hypoxia-inducible complex (HIF-1) to a hypoxia-responsive process requires protein synthesis (5Semenza G.L. Wang G.L. Mol. Cell. Biol. 1992; 12: 5447-5454Crossref PubMed Scopus (2174) Google Scholar) and is also dependent on as it is by several inhibitors (16Salceda S. Beck I. Srinivas V. Caro J. Kidney Int. 1997; 51: 556-559Abstract Full Text PDF PubMed Scopus (59) Google Scholar, G.L. Jiang B.-H. Semenza G.L. Biochem. Biophys. Res. Commun. 1995; PubMed Scopus Google Scholar). The protein components of this complex were recently and characterized as to the PAS of the basic helix-loop-helix of transcription factors (11Wang G.L. Jiang B.-H. Rue E.A. Semenza G.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5510-5514Crossref PubMed Scopus (4995) Google Scholar). of the protein HIF-1β, is the already known ARNT, the of the aryl hydrocarbon receptor protein. ARNT protein is constitutively expressed in normal cells and its level is not by hypoxic conditions. the the other HIF-1α, a new member of that family, is not expressed in normoxic cells, but accumulates rapidly under hypoxic conditions. Since HIF-1α mRNA is constitutively present in normoxic cells, the lack of HIF-1α protein is the of a lack of translation of the mRNA or the of a rapid degradation of the protein. A recent by al. (15Huang L.E. Arany Z. Livingston D.M. Bunn H.F. J. Biol. Chem. 1996; 271: 32253-32259Abstract Full Text Full Text PDF PubMed Scopus (1018) Google Scholar) that the of the HIF-1α protein is in normoxic conditions and is prolonged hypoxic The mechanisms for the rapid degradation of the protein were not Control of gene expression by regulated of transcription factors has been recently described to be an and mechanism of gene transcription (reviewed in Ref. H.L. Biol. 1996; PubMed Scopus Google Scholar). all transcription factors are degraded as of the of the stability of factors is of control at a very rapid rate and has the other of its cells on the and the proteasome for the degradation of for degradation are modified by the of of a basic to specific (reviewed in Ref. W. Biochem. Sci. 1996; Full Text PDF PubMed Scopus Google Scholar). The process requires several the one utilizing E1, an enzyme that a thiol the of regulated in the of HIF-1α, a series of inhibitors with proteolytic enzyme The cells were by hypoxia and the effect of the inhibitors on the rate of degradation of the HIF-1 complex was by shift These that controlled was involved in HIF-1 formation and that it was mediated by the proteasome it was by a highly specific inhibitor of the proteasome L. L. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). No significant effect was with the use of inhibitors or with inhibitors of enzyme the proteolytic system The lack of involvement of this last was by the use of a stably transfected cell line the protein J. T. G. Res. 1997; Google Scholar), which HIF-1 activation The of in the induction of HIF-1 was then studied in normoxic cells. These that induced HIF-1 complex formation in a to hypoxic confirmation of the involvement of the ubiquitin-proteasome system in the of HIF-1α protein levels and HIF-1 complex formation was obtained the use of a cell line containing a temperature-sensitive mutant of the ubiquitin-activating enzyme. These cell lines were in H. L. and to the degradation of (17Chowdary D.R. Dermody J.J. Jha K.K. Ozer H.L. Mol. Cell. Biol. 1994; 14: 1997-2003Crossref PubMed Scopus (266) Google Scholar). ts20 cells were cultured under normoxic conditions at the permissive temperature HIF-1 was whereas a shift to 39 °C a rapid accumulation of the complex. The HIF-1α protein contain several in other have been in degradation M. Biochem. Sci. 1996; Full Text PDF PubMed Scopus Google Scholar). However, the sequences that HIF-1α have not been is of to that proteasome induced HIF-1 complex formation in normoxic cells, not gene However, this is likely to a effect of these inhibitors lactacystin a in expression of the luciferase gene and the stimulation of its expression by cobalt, and hypoxia not is not HIF-1 complex formation is and for transcriptional activation. previously reported by Semenza et G.L. Jiang B.-H. R. A. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar) that of HIF-1α in normoxic conditions is to transcription of 1 its hypoxia response The mechanisms of oxygen sensing and the mechanisms by which hypoxia induces stabilization of the HIF-1α protein are currently unknown. The by several of an effect of on HIF-1 formation and Epo gene expression suggested that changes are likely to be involved in oxygen sensing signal Huang et al. (15Huang L.E. Arany Z. Livingston D.M. Bunn H.F. J. Biol. Chem. 1996; 271: 32253-32259Abstract Full Text Full Text PDF PubMed Scopus (1018) Google Scholar) recently reported that of the thiol reducing and hypoxia induced gene activation. The effect with the reducing thiol NMPG on HIF-1 formation and Epo gene activation in normoxic cells a that changes involved in the hypoxic The of the radicals that these signals and the mechanism of of the reducing are not A effect on HIF-1α the reducing already in their reduced changes could modify HIF-1α by the activity of and J. have that the induction of HIF-1 by NMPG could be by evidence suggest that radicals are in signal The of radicals in cells is strongly by the that 2-acetamidoacrylic the luciferase activity in cells under normoxic conditions. as as other have been described to with and superoxide and radicals M. 1993; PubMed Scopus Google Scholar). was recently reported that a J.M. M.A. A. J. Biochem. 1997; PubMed Scopus Google Scholar). have been described and in the of the mechanism for activation appears to on the of B.L. S. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). dependent changes of the degradation rate of by the proteasome system was recently reported to be dependent on the of M. 1997; PubMed Scopus Google Scholar). Since protein is a in HIF-1 the of in HIF-1α stabilization is a Dr. H. L. Ozer for the BALB/c 3T3 and ts20TG R cells and Dr. M. Institute, Medical University, for the stably transfected cell line the Drs. G. L. Semenza and C. A. Bradfield for the HIF-1α and Dr. S. Ōmura for of for for and R. for are to Dr. S. for the