Clioquinol, a Cu(II)/Zn(II) Chelator, Inhibits Both Ubiquitination and Asparagine Hydroxylation of Hypoxia-inducible Factor-1α, Leading to Expression of Vascular Endothelial Growth Factor and Erythropoietin in Normoxic Cells

We found that the Cu(II) and Zn(II)-specific chelator Clioquinol (10–50 μm) increased functional hypoxia-inducible factor 1α (HIF-1α) protein, leading to increased expression of its target genes, vascular endothelial growth factors and erythropoietin, in SH-SY5Y cells and HepG2 cells. Clioquinol inhibited ubiquitination of HIF-1α in a Cu(II)- and Zn(II)-dependent manner. It prevents FIH-1 from hydroxylating the asparagine residue (803) of HIF-1α in a Cu(II)- and Zn(II)-independent fashion. Therefore, it leads to the accumulation of HIF-1α that is prolyl but not asparaginyl hydroxylated. Consistent with this, co-immunoprecipitation assays showed that Clioquinol-induced HIF-1α interacted with cAMP-responsive element-binding protein in normoxic cells, implying that Clioquinol stabilizes the trans-active form of HIF-1α. Our results indicate that Clioquinol could be useful as an inducer of HIF-1α and its target genes in ischemic diseases. We found that the Cu(II) and Zn(II)-specific chelator Clioquinol (10–50 μm) increased functional hypoxia-inducible factor 1α (HIF-1α) protein, leading to increased expression of its target genes, vascular endothelial growth factors and erythropoietin, in SH-SY5Y cells and HepG2 cells. Clioquinol inhibited ubiquitination of HIF-1α in a Cu(II)- and Zn(II)-dependent manner. It prevents FIH-1 from hydroxylating the asparagine residue (803) of HIF-1α in a Cu(II)- and Zn(II)-independent fashion. Therefore, it leads to the accumulation of HIF-1α that is prolyl but not asparaginyl hydroxylated. Consistent with this, co-immunoprecipitation assays showed that Clioquinol-induced HIF-1α interacted with cAMP-responsive element-binding protein in normoxic cells, implying that Clioquinol stabilizes the trans-active form of HIF-1α. Our results indicate that Clioquinol could be useful as an inducer of HIF-1α and its target genes in ischemic diseases. Hypoxia-inducible factor 1 (HIF-1) 3The abbreviations used are: HIF-1α, hypoxia-inducible factor-1α; CBP, cAMP-responsive element-binding protein; EPO, erythropoietin; FIH-1, factor inhibiting HIF-1α; HRE, hypoxia-responsive element; ODD, oxygen-dependent degradation domain of HIF-1α; PHD, HIF-1α-specific prolyl hydroxylase; TPEN, N,N,N′,N′-tetrakis (2-pyridylmethyl) ethylenediamine; VHL, von Hippel-Lindau; VEGF, vascular endothelial growth factor; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; GST, glutathione S-transferase; E3, ubiquitin-protein isopeptide ligase. is a key regulator of hypoxic adaptation that functions by activating the transcription of several genes involved in angiogenesis, erythropoiesis, and glycolysis (1Masson N. Ratcliffe P.J. J. Cell Sci. 2003; 116: 3041-3049Crossref PubMed Scopus (189) Google Scholar, 2Seagroves T.N. Ryan H.E. Lu H. Wouters B.G. Knapp M. Thibault P. Laderoute K. Johnson R.S. Mol. Cell. Biol. 2001; 21: 3436-3444Crossref PubMed Scopus (495) Google Scholar). It consists of two subunits; HIF-1α is rapidly degraded under normoxic condition by the ubiquitin-proteasome system, whereas HIF-1β is stable. Under normoxic conditions the proline 564 and/or 402 residues of HIF-1α are hydroxylated by HIF-1α-specific prolyl-4 hydroxylases (PHDs), which need O2, α-ketoglutarate, vitamin C, and Fe(II) (3Bruick R.K. McKnight S.L. Science. 2001; 294: 1337-1340Crossref PubMed Scopus (2153) Google Scholar, 4Epstein A.C. Gleadle J.M. McNeill L.A. Hewitson K.S. O'Rourke J. Mole D.R. Mukherji M. Metzen E. Wilson M.I. Dhanda A. Tian Y.M. Masson N. Hamilton D.L. Jaakkola P. Barstead R. Hodgkin J. Maxwell P.H. Pugh C.W. Schofield C.J. Ratcliffe P.J. Cell. 2001; 107: 43-54Abstract Full Text Full Text PDF PubMed Scopus (2788) Google Scholar, 5Ivan M. Kondo K. Yang H. Kim W. Valiando J. Ohh M. Salic A. Asara J.M. Lane W.S. Kaelin W.G.J. Science. 2001; 292: 464-468Crossref PubMed Scopus (3981) Google Scholar, 6Jaakkola P. Mole D.R. Tian Y.M. Wilson M.I. Gielbert J. Gaskell S.J. Kriegsheim A. Hebestreit H.F. Mukherji M. Schofield C.J. Maxwell P.H. Pugh C.W. Ratcliffe P.J. Science. 2001; 292: 468-472Crossref PubMed Scopus (4572) Google Scholar). The hydroxylated prolines interact with von Hippel-Lindau (VHL) protein, a component of E3 ubiquitin ligase, and the HIF-1α is ubiquitinated by the VCB E3 ubiquitin-ligase complex, consisting of VHL protein, ElonginB, ElonginC, Cul2, and Rbx1 (7Iwai K. Yamanaka K. Kamura T. Minato N. Conaway R.C. Conaway J.W. Klausner D. Pause A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2436-12441Crossref Scopus (432) Google Scholar, 8Kamura T. Koepp D.M. Conrad M.N. Skowyra D. Moreland R.J. Iliopoulos O. Lane W.S. Kaelin Jr., W.G. Elledge S.J. Conaway R.C. Harper J.W. Conaway J.W. Science. 1999; 284: 657-661Crossref PubMed Scopus (680) Google Scholar). In hypoxic conditions, proline hydroxylation decreases and HIF-1α accumulates. Oxygen molecules inhibit not only the stabilization of HIF-1α but also its transactivation activity, because the protein, factor-inhibiting HIF-1α (FIH-1), catalyzes hydroxylation of its asparagine residue using O2, α-ketoglutarate, vitamin C, and Fe(II). Hydroxylation of this asparagine residue in the transactivation domain of HIF-1α prevents it from recruiting its coactivator, cAMP-response element-binding protein (CBP) (3Bruick R.K. McKnight S.L. Science. 2001; 294: 1337-1340Crossref PubMed Scopus (2153) Google Scholar, 9Mahon P.C. Hirota K. Semenza G.L. Genes Dev. 2001; 15: 2675-2686Crossref PubMed Scopus (1168) Google Scholar). Thus, activation of HIF-1α is censored by two systems, proline hydroxylation and asparagine hydroxylation. HIF-1α and its targets, such as EPO and VEGF, are being evaluated as therapeutic agents for cerebral and myocardial infarctions, and a small lipophilic HIF-1α-activating compound is being sought as a treatment for these diseases. However, to generate fully functional HIF-1α, a putative HIF-1α activator should suppress both proline hydroxylation-dependent ubiquitination and asparagine hydroxylation. We showed previously that the zinc chelator N,N,N′,N′-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN) enhances the activity of PHD2 but that the level of HIF-1α protein does not fall because TPEN also inhibits its ubiquitination. Because TPEN does not prevent FIH-1 from hydroxylating the asparagine residue of HIF-1α, it leads the accumulation of nonfunctional HIF-1α (10Choi S.M. Choi K.-O. Lee N. Oh M. Park H. Biochem. Biophys. Res. Commun. 2006; 343: 1002-1008Crossref PubMed Scopus (16) Google Scholar, 11Choi K.-O. Lee T. Lee N. Kim J. Yang E. Yoon J. Kim J. Lee T. Park H. Mol. Pharmacol. 2005; 68: 1803-1809Crossref PubMed Scopus (42) Google Scholar). Here, we report that another zinc chelator, Clioquinol, which has relatively low affinity but high selectivity for Zn(II) and Cu(II), has a different effect on the activity of HIF-1α. Both TPEN and Clioquinol inhibit ubiquitination of HIF-1α and cause its accumulation. However, in contrast to TPEN, Clioquinol prevents FIH-1 from hydroxylating HIF-1α. It therefore stabilizes functional HIF-1α, leading to expression of its target genes in normoxic cells. Clioquinol has been used in Alzheimer, Parkinson, and Huntington diseases as a Cu(II)- and Zn(II) chelator that reverses Zn(II)- or Cu(II)-induced metalloprotein precipitation (12Raman B. Ban T. Yamaguchi K. Sakai M. Kawai T. Naiki H. Goto Y. J. Biol. Chem. 2005; 280: 16157-16162Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). It was extensively used as an antibiotic in the mid-1900s but then withdrawn because it caused subacute myelo-optic neuropathy in Japan (13Cherny R.A. Atwood C.S. Xilinas M.E. Gray D.N. Jones W.D. McLean C.A. Barnham K.J. Volitakis I. Fraser F.W. Kim Y. Huang X. Goldstein L.E. Moir R.D. Lim J.T. Beyreuther K. Zheng H. Tanzi R.E. Masters C.L. Bush A.I. Neuron. 2001; 30: 665-676Abstract Full Text Full Text PDF PubMed Scopus (1342) Google Scholar, 14Kaur D. Yantiri F. Rajagopalan S. Kumar J. Mo J. Boonplueang R. Viswanath V. Jacobs R. Yang L. Beal M.F. DiMonte D. Volitaskis I. Ellerby L. Cherny R.A. Bush A.I. Andersen J.K. Neuron. 2003; 37: 899-909Abstract Full Text Full Text PDF PubMed Scopus (576) Google Scholar, 15Nguyen T. Hamby A. Massa S.M. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 11840-11845Crossref PubMed Scopus (170) Google Scholar). Here, we report that Clioquinol inhibits both ubiquitination and asparagine hydroxylation of HIF-1α and leads to activation of HIF-1α target genes. Cells, cDNAs, and Reagents—Human HepG2 hepatoma cells (ATCC HB-8065) and human SH-SY5Y neuroblastoma cells (ATCC CRL-2266) were purchased from the American Type Culture Collection and maintained as recommended. The cells were made hypoxic by incubation in hypoxic incubator (Model 1029; Forma Scientific, Inc.) at 37 °C. We used HIF-1α (U22431) and FIH-1(AF395830) human cDNAs as expression vectors as well as in transfection assays and in vitro transcription and translation experiments. The p(HRE)4-luc reporter plasmid contained four copies of the erythropoietin hypoxia-responsive element (nucleotides 3449–3470) (16Hur E. Chang K.Y. Lee E. Lee S. Park H. Mol. Pharmacol. 2001; 59: 1216-1224Crossref PubMed Scopus (115) Google Scholar). Anti-HIF-1α was obtained from BD Biosciences. We obtained MG132, TPEN, and Clioquinol from Calbiochem and all other chemicals from Sigma. Culture medium was purchased from Invitrogen and fetal bovine serum from BioWhittaker. Other chemicals were from Sigma. Measurement of PHD Activity by a VHL Pulldown Assay—The human PHD2 gene (identical to AJ310543) was cloned into the pET21b His2 (+) vector and overexpressed in Escherichia coli as histidine-tagged fused proteins and purified by nickel-affinity chromatography (11Choi K.-O. Lee T. Lee N. Kim J. Yang E. Yoon J. Kim J. Lee T. Park H. Mol. Pharmacol. 2005; 68: 1803-1809Crossref PubMed Scopus (42) Google Scholar). The in vitro VHL pulldown assay was performed as described by Jaakkola et al. (6Jaakkola P. Mole D.R. Tian Y.M. Wilson M.I. Gielbert J. Gaskell S.J. Kriegsheim A. Hebestreit H.F. Mukherji M. Schofield C.J. Maxwell P.H. Pugh C.W. Ratcliffe P.J. Science. 2001; 292: 468-472Crossref PubMed Scopus (4572) Google Scholar). Briefly, [35S]methionine-labeled VHL protein was synthesized by in vitro transcription and translation using the pcDNA3.1/hygro-VHL plasmid, according to the instruction manual (catalogue number L1170; Promega). GST-ODD (amino acids 401–603 of human HIF-1α) was expressed in E. coli and purified with glutathione-uniflow resin according to the instruction manual (catalogue number 8912-1; BD Biosciences Clontech). Resin-bound GST-ODD (200 μg of protein/∼80 μl of resin volume) was incubated in the presence of 2 mm ascorbic acid, 100 μm FeCl2, and 5 mm α-ketoglutarate with 1.5-3 μg of PHD2 in 200 μl of NETN buffer (20 mm Tris, pH 8.0, 100 mm NaCl, 1 mm EDTA, 0.5% Nonidet P-40, 1 mm phenylmethylsulfonyl fluoride) with mild agitation for 90 min at 30 °C. The reaction was and with of NETN Resin-bound GST-ODD was with μl of VHL in μl of buffer mm NaCl, mm pH 8.0, 0.5% Nonidet mild agitation at for 2 the resin was with 1 of NETN and proteins were in by and by The of was by the GST-ODD with In cells were with buffer (20 mm pH 5 mm mm 1 mm 2 2 mm phenylmethylsulfonyl the the cells were in a and the was at for min at to and of the were at °C. assays were at 30 for min in a of μl 2 μl of human μl of μg of μl of (20 mm Tris, pH mm mm mm μl of 5 ubiquitin and μl of μm ubiquitin buffer was and the reaction were by and M.E. Masson N. Mole D.R. Jaakkola P. Chang Pugh C.W. Ratcliffe P.J. Maxwell P.H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Measurement of FIH-1 human FIH-1 gene was cloned into vector and FIH-1 was overexpressed in E. coli as a histidine-tagged protein and purified by nickel-affinity The protein was purified by chromatography and by FIH-1 activity was by pulldown assays as described previously (10Choi S.M. Choi K.-O. Lee N. Oh M. Park H. Biochem. Biophys. Res. Commun. 2006; 343: 1002-1008Crossref PubMed Scopus (16) Google Scholar, Huang L.E. R. S. M. H.F. D.M. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). FIH-1 activity was by hydroxylation of Hydroxylation of was by (amino acids with an for another assay H. and E. was used for the FIH-1 The was incubated at a of μm with μg of FIH-1 in mm pH 5 mm and mm 100 μm α-ketoglutarate and μm ascorbic in a of incubation for 2 at were with The was from the with in by with in The was to a and were performed with a cells were to on and with as for in normoxic or hypoxic were as previously described (6Jaakkola P. Mole D.R. Tian Y.M. Wilson M.I. Gielbert J. Gaskell S.J. Kriegsheim A. Hebestreit H.F. Mukherji M. Schofield C.J. Maxwell P.H. Pugh C.W. Ratcliffe P.J. Science. 2001; 292: 468-472Crossref PubMed Scopus (4572) Google Scholar). of were by with 1 μg of and μl of 0.5% protein for 30 min at °C. The were with 1 μg of of μl of 0.5% protein were at °C. The were four with and in were then for 5 min and on and the proteins were to by proteins were with HIF-1α and/or and by according to the with or with as from protein on were with a (Model Clioquinol HIF-1α hepatoma HepG2 cells and neuroblastoma SH-SY5Y cells were to the TPEN and Clioquinol in both normoxic and hypoxic conditions, and of HIF-1α protein were by Both TPEN and Clioquinol increased the of HIF-1α in normoxic cells The in that of Zn(II) and Cu(II) the effect of Clioquinol, that it stabilizes HIF-1α by and Cu(II) R.A. J.T. McLean C.A. Huang X. Atwood C.S. Beyreuther K. Tanzi R.E. Masters C.L. Bush A.I. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, D.R. J. Sci. Full Text PDF PubMed Scopus Google Scholar). of Fe(II) to the effect of Clioquinol, that Fe(II) is not involved in this stabilization to expression of HIF-1α target genes we the expression of by In the Clioquinol but not TPEN increased expression of in normoxic cells whereas TPEN the expression of in SH-SY5Y cells. Clioquinol, but not TPEN, expression of under the of hypoxia-responsive in normoxic cells whereas TPEN expression in hypoxic cells the HIF-1α to the in the and transcription of target genes such as and Thus, the two both HIF-1α protein but in on transactivation of HIF-1α. of Clioquinol on the Activity of PHD2 and the of for this effect of Clioquinol we it the activity of We the hydroxylation activity of PHD2 by of VHL by the domain (amino acids of human HIF-1α protein, because the of VHL with HIF-1α on hydroxylation of the PHD2 was expressed with a in E. coli and purified as described previously (11Choi K.-O. Lee T. Lee N. Kim J. Yang E. Yoon J. Kim J. Lee T. Park H. Mol. Pharmacol. 2005; 68: 1803-1809Crossref PubMed Scopus (42) Google Scholar). We the purified PHD2 with Clioquinol or TPEN and its activity by VHL pulldown TPEN increased the activity of PHD2 (10Choi S.M. Choi K.-O. Lee N. Oh M. Park H. Biochem. Biophys. Res. Commun. 2006; 343: 1002-1008Crossref PubMed Scopus (16) Google whereas Clioquinol that the two zinc in effect on the activity of PHD2 We also for in the ubiquitination of HIF-1α in the presence of We synthesized HIF-1α and it to in vitro ubiquitination in the presence of ubiquitin and the of cells. The of high HIF-1α in that the HIF-1α was ubiquitinated in Clioquinol this ubiquitination. Anti-HIF-1α high HIF-1α protein in to the MG132, which inhibits the the degradation of HIF-1α in normoxic We high HIF-1α in cells to Clioquinol or TPEN that Clioquinol the ubiquitination of HIF-1α, it to in normoxic cells in with the results of the in vitro ubiquitination of Clioquinol on the Activity of of HIF-1α is inhibited by hydroxylation of its asparagine residue by an asparagine FIH-1 P.C. Hirota K. Semenza G.L. Genes Dev. 2001; 15: 2675-2686Crossref PubMed Scopus (1168) Google Scholar, R.K. J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, D. Science. PubMed Scopus Google Scholar). The that the HIF-1α in the presence of Clioquinol is to expression of its target genes that Clioquinol inhibits this hydroxylation. Because hydroxylation inhibits the of HIF-1α with its coactivator, CBP, HIF-1α is FIH-1 activity decreases S.J. F. D.M. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). HIF-1α was incubated with purified FIH-1 (10Choi S.M. Choi K.-O. Lee N. Oh M. Park H. Biochem. Biophys. Res. Commun. 2006; 343: 1002-1008Crossref PubMed Scopus (16) Google Scholar). HIF-1α interacted with protein, whereas HIF-1α to 1 and 1 Because FIH-1 the asparagine residue of HIF-1α, of to the transactivation domain of HIF-1α, the activity of FIH-1 is to the HIF-1α and We the effect of Clioquinol on FIH-1 In the presence of Clioquinol and 5 and and the activity of FIH-1 was that the HIF-1α and whereas TPEN to FIH-1 activity (10Choi S.M. Choi K.-O. Lee N. Oh M. Park H. Biochem. Biophys. Res. Commun. 2006; 343: 1002-1008Crossref PubMed Scopus (16) Google Scholar). The Clioquinol and TPEN the that Clioquinol, but not TPEN, the expression of HIF-1α target genes the zinc domain is in the of CBP, Clioquinol TPEN the and HIF-1α 1 and Our results therefore that the HIF-1α in the presence of Clioquinol is to its because its but not its residues are hydroxylated. TPEN to inhibit the activity of FIH-1, nonfunctional HIF-1α. the effect of Clioquinol on FIH-1 on Zn(II) or Cu(II), we these to the FIH-1 hydroxylation reaction with Both Cu(II) and Zn(II) to the effect of Clioquinol, that Clioquinol inhibits the asparagine hydroxylation activity of FIH-1 in a Cu(II)- and Zn(II)-independent and is with the that the other Cu(II) and Zn(II) chelator, TPEN, to inhibit the activity of We the effect of Clioquinol on HIF-1α at to its effect is for asparagine hydroxylation of HIF-1α. The in which asparagine is with its to interact with FIH-1 was incubated with it to prevent from recruiting CBP, and Clioquinol not the of to of the presence of FIH-1 that Clioquinol the of to HIF-1α by FIH-1 from hydroxylating the activity of the FIH-1 by hydroxylation of by its with CBP, we incubated HIF-1α (amino acids with purified FIH-1 and the in of the treatment with FIH-1 the a to an of of and that the FIH-1 of HIF-1α. In the was incubated with FIH-1 and Clioquinol its not and that Clioquinol inhibits the activity of of Clioquinol on by that Clioquinol inhibits asparagine hydroxylation of HIF-1α that it the transactivation activity of HIF-1α, as its with its assays showed that HIF-1α with in both cells and hypoxic cells, but not in or cells, that the HIF-1α to or TPEN from that in cells in of its to interact with TPEN Clioquinol with the HIF-1α and in vitro has the zinc domain for with HIF-1α. that the domain is not by the two at the that Clioquinol the transactivation activity of HIF-1α in we used a reporter plasmid the gene under the of the We a reporter plasmid into HepG2 cells with plasmid which HIF-1α to the domain of the protein (amino acids Because only the protein is to to the the reporter gene is only HIF-1α has Clioquinol increased the activity of HIF-1α in normoxic cells We the of and EPO in SH-SY5Y cells by The results in that Clioquinol the expression of both EPO and in SH-SY5Y cells. We that both TPEN and Clioquinol HIF-1α protein in normoxic cells by its ubiquitination. The that the of Cu(II) and Zn(II) the of Clioquinol that it stabilizes HIF-1α in a Cu(II)- and Zn(II)-specific manner. of HIF-1α is but not to expression of its target genes (10Choi S.M. Choi K.-O. Lee N. Oh M. Park H. Biochem. Biophys. Res. Commun. 2006; 343: 1002-1008Crossref PubMed Scopus (16) Google Scholar, E. Chang K.Y. Lee E. Lee S. Park H. Mol. Pharmacol. 2001; 59: 1216-1224Crossref PubMed Scopus (115) Google Scholar). transactivation activity, the asparagine of the HIF-1α to be not hydroxylated by FIH-1 as to be to interact with We found that the two in effect on the transactivation activity of HIF-1α. Clioquinol, but not TPEN, inhibited the activity of We that Clioquinol does not inhibit the activity of FIH-1 by for the the other Zn(II) and Cu(II) chelator TPEN to inhibit the activity of FIH-1, of Cu(II) or to the effect of Clioquinol on the asparagine hydroxylation of HIF-1α, and Zn(II) inhibits the activity of FIH-1 in vitro μm) K.S. McNeill L.A. Tian Y.M. J.M. S. Gleadle J.M. Ratcliffe P.J. Pugh C.W. Schofield C.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). HIF-1α to interact with CBP, not because TPEN with its with but because it to inhibit its asparagine hydroxylation by In Clioquinol-induced HIF-1α does interact with CBP, because it inhibits FIH-1, asparagine hydroxylation of HIF-1α. Therefore, Clioquinol has a effect both ubiquitination and asparagine hydroxylation of HIF-1α. Both relatively high for Zn(II) and Cu(II) and other The of TPEN are: Cu(II), of Clioquinol are: Cu(II), R.A. J.T. McLean C.A. Huang X. Atwood C.S. Beyreuther K. Tanzi R.E. Masters C.L. Bush A.I. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, D.R. J. Sci. Full Text PDF PubMed Scopus Google Scholar). Both TPEN and Clioquinol Zn(II)- or Cu(II)-induced metalloprotein precipitation (12Raman B. Ban T. Yamaguchi K. Sakai M. Kawai T. Naiki H. Goto Y. J. Biol. Chem. 2005; 280: 16157-16162Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, C.J. R. Volitakis I. Cherny R.A. Beyreuther K. Masters C.L. Bush A.I. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Because Clioquinol is has a low and the it is being as a compound that decreases of in Alzheimer, Parkinson, and Huntington diseases and the to Clioquinol was extensively used as an antibiotic in the mid-1900s but was then withdrawn because it caused subacute myelo-optic neuropathy (13Cherny R.A. Atwood C.S. Xilinas M.E. Gray D.N. Jones W.D. McLean C.A. Barnham K.J. Volitakis I. Fraser F.W. Kim Y. Huang X. Goldstein L.E. Moir R.D. Lim J.T. Beyreuther K. Zheng H. Tanzi R.E. Masters C.L. Bush A.I. Neuron. 2001; 30: 665-676Abstract Full Text Full Text PDF PubMed Scopus (1342) Google Scholar, 14Kaur D. Yantiri F. Rajagopalan S. Kumar J. Mo J. Boonplueang R. Viswanath V. Jacobs R. Yang L. Beal M.F. DiMonte D. Volitaskis I. Ellerby L. Cherny R.A. Bush A.I. Andersen J.K. Neuron. 2003; 37: 899-909Abstract Full Text Full Text PDF PubMed Scopus (576) Google Scholar, 15Nguyen T. Hamby A. Massa S.M. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 11840-11845Crossref PubMed Scopus (170) Google Scholar). In a of which an Clioquinol both and serum of Full Text Full Text PDF PubMed Scopus Google Scholar). in with showed that Clioquinol and and of subacute myelo-optic neuropathy were C.W. Bush A.I. A. S. M. L. L. Cherny R. A. D. Volitakis I. Xilinas M. D. S. Beyreuther K. Tanzi R.E. Masters C.L. 2003; PubMed Scopus Google Scholar). The and of of other Clioquinol also in several human B. H. Res. 2005; PubMed Scopus Google Scholar). is a regulator that ischemic by several genes for angiogenesis, erythropoiesis, and of HIF-1α also the and of G.L. 2003; PubMed Scopus Google Scholar, L. X. M. A. D. Y. Res. 2005; PubMed Scopus Google Scholar). In it has on ischemic to the and that a low activator of HIF-1α and its target genes could a treatment for and myocardial R. R.C. Res. 2006; PubMed Scopus Google Scholar, A. L. S. S.M. Cherny R.A. Volitakis I. Bush A.I. I. T. V. J. Biol. Chem. 2005; 280: Full Text Full Text PDF PubMed Scopus Google Scholar). The of are by its target genes, the of which are EPO and VEGF, which are of the effect of hypoxic A. M. H. M. M. M. PubMed Scopus Google Scholar, L. R. J. A. P. P. M. A. M. Proc. Natl. Acad. Sci. U. S. A. 2003; PubMed Scopus Google Scholar, D.M. 2005; PubMed Scopus Google Scholar, M. 2003; PubMed Scopus Google Scholar). of to in of and the by this was in K. Lu H. Semenza G.L. 2003; PubMed Scopus Google Scholar). EPO increased functional and in to The by hypoxic is on HIF-1α and EPO L. R. J. A. P. P. M. A. M. Proc. Natl. Acad. Sci. U. S. A. 2003; PubMed Scopus Google Scholar). EPO and its are expressed in the and to from ischemic M. S. L.A. M. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, H. H. H. S. M. PubMed Scopus (115) Google Scholar). In a of EPO in with of and H. M. L. P. M. N. S. F. M. W. E. M. O. M. L. H. A. M. Mol. PubMed Google Scholar). In to EPO, of the in of myocardial and its activity a activity on and J.M. J.M. PubMed Scopus Google and of cells into cerebral W. Y. Y. Y. Y. Yang 2005; PubMed Scopus Google Scholar). However, of or EPO a Our that the lipophilic and small compound Clioquinol HIF-1α and its target genes, in and EPO, in normoxic cells that Clioquinol and ischemic and other diseases.