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

Abstract

Hypoxia-inducible factor 1 (HIF-1) 3 is a key regulator of hypoxic adaptation that functions by activating the transcription of several genes involved in angiogenesis, erythropoiesis, and glycolysis (1, 2). It consists of two subunits; HIF-1␣ is rapidly degraded under normoxic condition by the ubiquitin-proteasome system, whereas HIF-1␤ is stable. Under normoxic conditions the proline 564 and/or 402 residues of HIF-1␣ are hydroxylated by HIF-1␣-specific prolyl-4 hydroxylases (PHDs), which need O 2 , ␣-ketoglutarate, vitamin C, and Fe(II) (3-6). The hydroxylated prolines interact with von Hippel-Lindau (VHL) protein, a component of E3 ubiquitin ligase, and the HIF-1␣ is ubiquitinated by the VCB E3 ubiquitin-ligase complex, consisting of VHL protein, ElonginB, ElonginC, Cul2, and Rbx1 (7,8). In hypoxic conditions, proline hydroxylation decreases and HIF-1␣ accumulates. Oxygen molecules inhibit not only the stabilization of HIF-1␣ but also its transactivation activity, because the protein, factor-inhibiting HIF-1␣ (FIH-1), catalyzes hydroxylation of its asparagine residue using O 2 , ␣-ketoglutarate, vitamin C, and Fe(II). Hydroxylation of this asparagine residue in the transactivation domain of HIF-1␣ prevents it from recruiting its coactivator, cAMP-response element-binding protein (CBP) (3, 9).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). It

Biometals and Their Therapeutic Implications in Alzheimer's Disease

“…Although it was initially considered a chelator [198][199][200][201], it has more recently been characterized as a copper/zinc ionophore, which functions to redistribute these metals into cells [196,[202][203][204][205][206]. Clioquinol is still considered a moderate iron chelator as it has been shown to lower iron levels in animal models of iron overload [64,148,[207][208][209][210], and has not been shown to redistribute iron into cells using ionophore assays.…”

Glucocorticoid Protection of Oligodendrocytes against Excitotoxin Involving Hypoxia-Inducible Factor-1 in a Cell-Type-Specific Manner

“…4C,D). Activation of HIF-1␣ under normoxia has been demonstrated previously in selected experimental paradigms (Choi et al, 2006;Liu et al, 2007) with RACK1 (the receptor for activated C kinase 1) and FIH-1 (factor inhibiting HIF) playing an important role. It remains to be determined whether these factors are involved in the GR activation of HIF-1␣.…”

The novel peptide F29 facilitates the DNA-binding ability of hypoxia-inducible factor-1α

“…Clioquinol is also an HIF-1α activator and leads to the accumulation of the trans-active form of HIF-1α by inhibiting both FIH-1 and HIF-1α ubiquitination (6). Since these chemicals target upstream regulators instead of HIF-1α directly, we should be concerned with potential side effects.…”

“…Cells were exposed to hypoxia (0.1% O2) using an anaerobic incubator (Model 1029, Forma Scientific, Inc.) or chemical hypoxic mimicker CoCl2 (Sigma Co.) (6). The MATCHMAKER random peptide library (Cat# NL4000AA) and GAL4 two-hybrid system 3 (Cat# K1612-1) were purchased from Clontech (Takara Bio Inc., Palo Alto, CA, USA).…”

“…We have identified small compounds that activate HIF-1α, such as clioquinol, SNAP ((±)-S-nitroso-N-acetylpenicillamine) and baicalein (6)(7)(8). These compounds inhibit both asparaginyl hydroxylation and proline hydroxylation-dependent ubiquitination, leading to the accumulation of trans-active HIF-1α even in normoxic conditions.…”