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.

Thus, activation of HIF-1␣ is censored by two systems, proline hydroxylation and asparagine hydroxylation. HIF-1␣ and its targets, such as EPO and VEGF, are being evaluated as therapeutic agents for cerebral and myocardial infarctions, and a small lipophilic HIF-1␣-activating compound is being sought as a treatment for these diseases. However, to generate fully functional HIF-1␣, a putative HIF-1␣ activator should suppress both proline hydroxylation-dependent ubiquitination and asparagine hydroxylation.
We showed previously that the zinc chelator N,N,NЈ,NЈ-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN) enhances the activity of PHD2 but that the level of HIF-1␣ protein does not fall because TPEN also inhibits its ubiquitination. Because TPEN does not prevent FIH-1 from hydroxylating the asparagine residue of HIF-1␣, it leads the accumulation of nonfunctional HIF-1␣ (10,11). 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-1␣. Both TPEN and Clioquinol inhibit ubiquitination of HIF-1␣ and cause its accumulation. However, in contrast to TPEN, Clioquinol prevents FIH-1 from hydroxylating HIF-1␣. It therefore stabilizes functional HIF-1␣, 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 (12 was extensively used as an antibiotic in the mid-1900s but then withdrawn because it caused subacute myelo-optic neuropathy in Japan (13)(14)(15). Here, we report that Clioquinol inhibits both ubiquitination and asparagine hydroxylation of HIF-1␣ and leads to activation of HIF-1␣ target genes.

EXPERIMENTAL PROCEDURES
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-1␣ (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) (16). Anti-HIF-1␣ 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 (ϩ) vector and overexpressed in Escherichia coli as histidine-tagged fused proteins and purified by nickel-affinity chromatography (11). The in vitro VHL pulldown assay was performed as described by Jaakkola et al. (6). Briefly, [ 35 S]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-1␣) 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 g of protein/ϳ80 l of resin volume) was incubated in the presence of 2 mM ascorbic acid, 100 M FeCl 2 , and 5 mM ␣-ketoglutarate with 1.5-3 g of PHD2 in 200 l 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 mixture was centrifuged and washed three times with 10 volumes of NETN buffer. Resin-bound GST-ODD was mixed with 10 l of 35 S-labeled VHL in 500 l of EBC buffer (120 mM NaCl, 50 mM Tris-HCl, pH 8.0, 0.5% (v/v) Nonidet P-40). After mild agitation at 4°C for 2 h, the resin was washed three times with 1 ml of NETN buffer, and proteins were eluted in 3ϫ SDS sample buffer, fractionated by 12% SDS-PAGE, and detected by autoradiography. The amount of each sample loaded was monitored by staining the GST-ODD with Coomassie Blue.
In Vitro Ubiquitination-HeLa cells were washed twice with cold hypotonic extraction buffer (20 mM Tris-HCl, pH 7.5, 5 mM KCl, 1.5 mM MgCl 2 , 1 mM dithiothreitol, 2 g/ml aprotinin, 2 g/ml leupeptin, 0.2 mM phenylmethylsulfonyl fluoride). After removing the buffer, the cells were disrupted in a Dounce homogenizer, and the crude extract was centrifuged at 10,000 ϫ g for 10 min at 4°C to remove cell debris and nuclei. Aliquots of the supernatant (S-10 fraction) were stored at Ϫ70°C. Ubiquitination assays were carried out at 30°C for 270 min in a total volume of 40 l containing 2 l of 35 S-labeled human HIF-1␣-programmed reticulocyte lysate, 27 l of S-10 extract (50 g of protein), 4 l of 10 ϫ ATP-regenerating system (20 mM Tris, pH 7.5, 10 mM ATP, 10 mM magnesium acetate, 300 mM creatine phosphate, 0.5 mg/ml creatine phos- phokinase), 4 l of 5 mg/ml ubiquitin (Sigma), and 0.83 l of 150 M ubiquitin aldehyde (Sigma). SDS sample buffer was added, and the reaction products were analyzed by 6% SDS-PAGE and autoradiography (17).
Measurement of FIH-1 Activity-The human FIH-1 gene (AF395830) was cloned into pET28a vector (Novagen), and FIH-1 was overexpressed in E. coli as a histidine-tagged fusion protein and purified by nickel-affinity chromatography. The fusion protein was further purified by gel filtration chromatography (Hi-Load Superdex 200) and concentrated by ultrafiltration. FIH-1 activity was measured by GST-CBP pulldown assays as described previously (10,18). Alternatively FIH-1 activity was measured by Asn hydroxylation of F-HIF-1␣ peptide. Hydroxylation of peptide was measured by mass spectrophotometric analysis. F-HIF-1␣ (amino acids 788 -822) peptide (fluorescein isothiocyanate-aca-DESGLPQLTSYDCEVNAPIQGSRNLLQGEELLRAL) containing fluorescein conjugated with an N-terminal-inserted aminocaproic acid (aca) linker (AnyGen, KwangJu, Korea) developed for another assay 4 was used for the FIH-1 reaction. The peptide was incubated at a final concentration of 4 M with 2.8 g of recombinant FIH-1 in 20 mM Tris buffer, pH 7.5, 5 mM KCl, and 1.5 mM MgCl 2 containing 100 M ␣-ketoglutarate and 400 M ascorbic acid in a total volume of 50 l. After incubation for 2 h at room temperature, excess salts were removed with ZipTip C18 (Millipore). The peptide was eluted from the tip with ␣-cyano-4-hydroxycinnamic acid in acetonitrile/water containing 0.1% trifluoroacetic acid (50:50, v/v) followed by extensive washing with 0.1% trifluoroacetic acid in water. The eluted peptide solution was transferred to a MALDI sample plate and MALDI-TOF measurements were performed with a Voyager analyzer (Applied Biosystems).
Co-immunoprecipitation-HepG2 cells were grown to 80% confluence on 100-mm tissue culture plates and treated with drugs as indicated for 6 h in normoxic or hypoxic conditions. Whole cell extracts were prepared as previously described (6). For immunoprecipitation, 200-g samples of whole cell lysates were precleared by incubating with 1 g of anti-mouse IgG (Santa Cruz Biotechnology, Santa Cruz, CA) and 20 l of 0.5% ImmunoPure immobilized protein A/G gel (Pierce) for 30 min at 4°C. The cleared extracts were mixed with 1 g of anti-CBP antibody (Santa Cruz Biotechnology). After addition of 15 l of 0.5% ImmunoPure immobilized protein A/G gel, they were rotated overnight at 4°C. The immunoprecipitates were pelleted, washed four times with phosphate-buffered saline, and resuspended in SDS sample buffer. They were then boiled for 5 min and run on 8% SDS-polyacrylamide gels, and the proteins were transferred to nitrocellulose membranes by semi-dry transfer (Trans-Blot S.D.; Bio-Rad). Co-immunoprecipitated proteins were reacted with anti-human HIF-1␣ antibody (BD Biosciences) and/or anti-CBP antibody and visualized by enhanced chemiluminescence according to the manufacturer's instructions (Pierce) with anti-mouse or rabbit Ig conjugated with horseradish peroxidase as secondary antibody. Weak signals from protein bands on Western blots were visualized with a luminescence image analyzer (Model LAS-3000; Fuji).

RESULTS
Clioquinol Stabilizes Functional HIF-1␣ Protein-Human hepatoma HepG2 cells and neuroblastoma SH-SY5Y cells were exposed to the cation chelators TPEN and Clioquinol in both normoxic and hypoxic (1% O 2 ) conditions, and levels of HIF-1␣ protein were measured by Western blotting. Both TPEN and Clioquinol dramatically increased the amount of HIF-1␣ in normoxic cells (Fig. 1A). The data in Fig. 1B   Cu(II) (19,20). Addition of Fe(II) failed to reverse the effect of Clioquinol, indicating that Fe(II) is not involved in stabilization.
To see whether this stabilization led to expression of HIF-1␣ target genes we measured the expression of VEGF by reverse transcriptase PCR. In the event, Clioquinol but not TPEN increased expression of VEGF in normoxic cells ( Fig. 2A), whereas TPEN reduced the hypoxia-induced expression of VEGF in SH-SY5Y cells. Similarly, Clioquinol, but not TPEN, induced expression of luciferase under the control of hypoxiaresponsive elements (HRE) in normoxic cells whereas TPEN decreased expression in hypoxic cells (Fig. 2B). Ultimately, the activated HIF-1␣ binds to the HRE located in the enhancer region and thus increases transcription of target genes such as VEGF and EPO. Thus, the two chelators both stabilized HIF-1␣ protein but differed in their effects on transactivation of HIF-1␣.
Effects of Clioquinol on the Activity of PHD2 and the Ubiquitination of HIF-1␣-To account for this effect of Clioquinol we tested whether it changed the activity of PHD2. We examined the hydroxylation activity of PHD2 by measuring capture of 35 S-labeled VHL by the ODD domain (amino acids 401-603) of human HIF-1␣ protein, because the interaction of VHL with HIF-1␣ depends on hydroxylation of the proline-402/564 residues. Human PHD2 was expressed with a histidine tag in E. coli and purified as described previously (11). We treated the purified full-length PHD2 with either Clioquinol or TPEN and measured its activity by VHL pulldown assays. TPEN increased the activity of PHD2 (10), whereas Clioquinol did not. This result demonstrates that the two zinc chelators differ in their effect on the activity of PHD2 (Fig. 3A).
We also looked for any change in the ubiquitination of HIF-1␣ in the presence of Clioquinol. We synthesized 35 S-labeled HIF-1␣ and subjected it to in vitro ubiquitination in the presence of ubiquitin, ubiquitin aldehyde, and the S-10 fraction of cells. The ladder-like appearance of high molecular weight HIF-1␣ in Fig. 3B confirms that the 35 S-labeled HIF-1␣ was ubiquitinated in vitro. Interestingly, Clioquinol repressed this ubiquitination.
Anti-HIF-1␣ antibody recognizes high molecular mass HIF-1␣ protein in cell lysates exposed to the proteasome inhibitor MG132, which specifically inhibits the 26S proteasome, thereby reducing the degradation of ubiquitin-conjugated HIF-1␣ even in normoxic conditions. We detected much less high molecular mass HIF-1␣ in cells exposed to either Clioquinol or TPEN (Fig. 3C). This observation shows that Clioquinol blocks the ubiquitination of HIF-1␣, thereby causing it to accumulate in normoxic cells in agreement with the results of the in vitro ubiquitination assay.
To test whether the inhibitory effect of Clioquinol on FIH-1 depends on Zn(II) or Cu(II), we added these cations to the FIH-1 hydroxylation reaction together with Clioquinol. Both Cu(II) and Zn(II) failed to reverse the inhibitory effect of Clioquinol, indicating that Clioquinol inhibits the asparagine hydroxylation activity of FIH-1 in a Cu(II)-and Zn(II)-independent manner (Fig. 4, C and D). This result is consistent with the finding that the other Cu(II) and Zn(II) chelator, TPEN, fails to inhibit the activity of FIH-1.
We tested the effect of Clioquinol on HIF-1␣ mutated at Asn-803 to see whether its effect is specific for asparagine 803 hydroxylation of HIF-1␣. The HIF-1␣-C(N803A) mutant, in which asparagine 803 is substituted with alanine, retains its ability to interact with GST-CBP. When FIH-1 was incubated with radiolabeled HIF-1␣-C(N803A) it failed to prevent HIF-1␣-C(N803A) from recruiting CBP, and Clioquinol did not enhance the recruitment of CBP to HIF-1␣-C(N803A) regardless of the presence of FIH-1 (Fig. 4E). This finding suggests that Clioquinol stimulates the recruitment of CBP to HIF-1␣ by preventing FIH-1 from hydroxylating Asn-803.
To determine the activity of the FIH-1 by measuring hydroxylation of Asn-803 rather than by assessing its interaction with CBP, we incubated HIF-1␣ peptide (amino acids 788 -822) with purified recombinant FIH-1 and determined the change in molecular mass of the peptide MALDI-TOF analysis. After treatment with FIH-1 the peptide gave a new MALDI-TOF peak corresponding to an increase of molecular weight of 16 (Fig. 5, A and B). This confirms that the recombinant FIH-1 hydroxylates Asn-803 of HIF-1␣. In contrast, when the peptide was incubated with FIH-1 and Clioquinol its molecular weight did not increase (Fig. 5, B and D). This observation confirms that Clioquinol inhibits the Asn-hydroxylating activity of FIH-1.
Effects of Clioquinol on Hypoxia-induced Transactivation by HIF-1␣-The fact that Clioquinol inhibits asparagine hydroxylation of HIF-1␣ implies that it increases the transactivation activity of HIF-1␣, defined as its interaction with its coactivator CBP. Co-immunoprecipitation assays showed that HIF-1␣ interacts with CBP in both Clioquinol-treated cells and hypoxic cells, but not in MG132-or TPEN-treated cells, indicating that the HIF-1␣ accumulated upon exposure to MG132 or TPEN differs from that in Clioquinol-treated cells in terms of its ability to interact with CBP (Fig. 6A). Interestingly, neither TPEN nor Clioquinol interferes with the interaction between HIF-1␣ and CBP in vitro (Fig. 6B), although CBP has the zinc finger CH1 domain required for interaction with HIF-1␣. This suggests that the CH1 domain is not affected by the two chelators at the concentrations used.
To confirm that Clioquinol increases the transactivation activity of HIF-1␣ in vivo, we used a Gal4-driven reporter plas- mid encoding the firefly luciferase gene under the control of the Gal4 binding site. We transfected a Gal4-driven reporter plasmid into HepG2 cells together with plasmid pGal4/HIF-1␣, which expresses HIF-1␣ linked to the DNA binding domain of the yeast Gal4 protein (amino acids 1-147). Because only the Gal4 fusion protein is able to bind to the Gal4 binding site, the reporter gene is transcribed only when HIF-1␣ has trans-activation activity. As expected, Clioquinol increased the transactivation activity of HIF-1␣ in normoxic cells (Fig. 6C). We examined the levels of VEGF and EPO mRNA in Clioquinoltreated SH-SY5Y cells by Northern analysis. The results in Fig.  6D confirm that Clioquinol strongly induces the expression of both EPO and VEGF in SH-SY5Y cells.

DISCUSSION
We have shown that both TPEN and Clioquinol stabilize HIF-1␣ protein in normoxic cells by blocking its ubiquitination. The fact that the addition of Cu(II) and Zn(II) reversed the effects of Clioquinol suggests that it stabilizes HIF-1␣ in a Cu(II)-and Zn(II)-specific manner. Stabilization of HIF-1␣ is necessary but not sufficient to induce expression of its target genes (10,16). For transactivation activity, the asparagine of the stabilized HIF-1␣ needs to be not hydroxylated by FIH-1 so as to be able to interact with CBP. We found that the two chelators differed in their effect on the transactivation activity of HIF-1␣. Clioquinol, but not TPEN, inhibited the activity of FIH-1. We suggest that Clioquinol does not inhibit the activity of FIH-1 by cation chelation for the following reasons: (i) the other Zn(II) and Cu(II) chelator TPEN fails to inhibit the activity of FIH-1, (ii) addition of Cu(II) or Zn (II) fails to reverse the inhibitory effect of Clioquinol on the asparagine hydroxylation of HIF-1␣, and (iii) Zn(II) rather inhibits the activity of recombinant FIH-1 in vitro (IC 50 , 10 M) (24). Accordingly, TPEN-induced HIF-1␣ fails to interact with CBP, not because TPEN interferes directly with its interaction with CBP but because it fails to inhibit its asparagine hydroxylation by FIH-1. In contrast, Clioquinol-induced HIF-1␣ does interact with CBP, because it inhibits FIH-1, thus blocking asparagine hydroxylation of HIF-1␣. Therefore, Clioquinol has a dual inhibitory effect blocking both ubiquitination and asparagine hydroxylation of HIF-1␣.  (19,20). Both TPEN and Clioquinol reverse Zn(II)-or Cu(II)-induced metalloprotein precipitation (12,25).
Because Clioquinol is hydrophobic, has a low general toxicity profile, and crosses the blood brain barrier, it is being reevaluated as a prototype metal-protein-attenuating compound that decreases deposits of metalloproteins in Alzheimer, Parkinson, and Huntington diseases and the oxidative stress due to them. Clioquinol was extensively used as an antibiotic in the mid-1900s but was then withdrawn because it caused subacute myelo-optic neuropathy (13)(14)(15). In a study of APP2576 transgenic mice, which have an Alzheimer disease-type neuropathy, Clioquinol reduced both amyloid ␤ plaques and serum levels of amyloid ␤ without systemic adverse effects (26). A recent phase II clinical trial in 36 patients with Alzheimer disease showed that Clioquinol slowed cognitive decline and decreased plasma amyloid ␤ concentrations, and no cases of subacute myelo-optic neuropathy were reported (27). The development and optimization of Clioquinol-like metal-protein-attenuating compounds requires careful study of other possible adverse effects. Clioquinol also causes apoptotic cell death in several human cancer cell lines (28).
HIF-1 is a master regulator that attenuates ischemic injury by inducing several genes required for angiogenesis, erythropoiesis, glycolysis, and vasodilation. Activation of HIF-1␣ also promotes the survival and progression of various cancers (29,30). In contrast, it has beneficial effects on ischemic injury to the heart and brain, suggesting that a low molecular weight activa- tor of HIF-1␣ and its target genes could provide a novel treatment for stroke and myocardial infarction (31,32). The beneficial effects of HIF-1 are mostly mediated by its target genes, the most prominent of which are EPO and VEGF, which are considered major mediators of the protective effect of hypoxic preconditioning (33)(34)(35)(36). Pre-exposure of wild-type mice to brief hypoxia resulted in protection of isolated hearts against ischemia-reperfusion injury 24 h later, and the cardiac protection induced by this preconditioning program was lost in HIF-1␣ ϩ/Ϫ mice (37). EPO administration increased functional recovery and decreased apoptosis in isolated hearts subjected to ischemia 24 h later. The cardiac protection induced by hypoxic preconditioning is critically dependent on HIF-1␣ and EPO (34). Interestingly, EPO and its receptor are expressed in the brain and contribute to neuroprotection from ischemic damage (38,39). In a first clinical trial of EPO in patients with acute stroke, EPO-treated patients had significantly reduced size of infarct and improved clinical outcome (40). In addition to EPO, administration of the angiogenic cytokine VEGF improves tissue perfusion via neovascularization in animal models of myocardial and limb ischemia. Besides its angiogenic activity VEGF had a novel protective activity on damaged neurons and glia (41), and transplantation of VEGF-transfected neural stem cells into rat brain provided neuroprotection after transient focal cerebral ischemia (42).
However, effective delivery of VEGF or EPO remains a challenge. Our finding that the lipophilic and less toxic small compound Clioquinol activates HIF-1␣ and its target genes, in particular VEGF and EPO, in normoxic cells suggests that Clioquinol might provide preconditioning protection against myocardial, neuronal, and limb ischemic injuries and other neurodegenerative diseases.