Clioquinol, a Cu(II)/Zn(II) chelator, inhibits both ubiquitination and asparagine hydroxylation of hypoxia-inducible factor-1alpha, 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 microM) increased functional hypoxia-inducible factor 1alpha (HIF-1alpha) 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-1alpha in a Cu(II)- and Zn(II)-dependent manner. It prevents FIH-1 from hydroxylating the asparagine residue (803) of HIF-1alpha in a Cu(II)- and Zn(II)-independent fashion. Therefore, it leads to the accumulation of HIF-1alpha that is prolyl but not asparaginyl hydroxylated. Consistent with this, co-immunoprecipitation assays showed that Clioquinol-induced HIF-1alpha interacted with cAMP-responsive element-binding protein in normoxic cells, implying that Clioquinol stabilizes the trans-active form of HIF-1alpha. Our results indicate that Clioquinol could be useful as an inducer of HIF-1alpha and its target genes in ischemic diseases.     

Coordination changes and auto-hydroxylation of FIH-1: uncoupled O2-activation in a human hypoxia sensor

Hypoxia sensing is the generic term for pO₂-sensing in humans and other higher organisms. These cellular responses to pO₂ are largely controlled by enzymes that belong to the Fe(II) α-ketoglutarate (αKG) dependent dioxygenase superfamily, including the human enzyme called the factor inhibiting HIF (FIH-1), which couples O₂-activation to the hydroxylation of the hypoxia inducible factor α (HIFα). Uncoupled O₂-activation by human FIH-1 was studied by exposing the resting form of FIH-1 (αKG + Fe)FIH-1, to air in the absence of HIFα. Uncoupling lead to two distinct enzyme oxidations, one a purple chromophore (λ_max = 583 nm) arising from enzyme auto-hydroxylation of Trp²⁹⁶, forming an Fe(III)-O-Trp²⁹⁶ chromophore [Y.-H. Chen, L.M. Comeaux, S.J. Eyles, M.J. Knapp, Chem. Commun. (2008), doi:10.1039/B809099H]; the other a yellow chromophore due to Fe(III) in the active site, which under some conditions also contained variable levels of an oxygenated surface residue (oxo)Met²⁷⁵. The kinetics of purple FIH-1 formation were independent of Fe(II) and αKG concentrations, however, product yield was saturable with increasing [αKG] and required excess Fe(II). Yellow FIH-1 was formed from (succinate + Fe)FIH-1, or by glycerol addition to (αKG + Fe)FIH-1, suggesting that glycerol could intercept the active oxidant from the FIH-1 active site and prevent hydroxylation. Both purple and yellow FIH-1 contained high-spin, rhombic Fe(III) centers, as shown by low temperature EPR. XAS indicated distorted octahedral Fe(III) geometries, with subtle differences in inner-shell ligands for yellow and purple FIH-1. EPR of Co(II)-substituted FIH-1 (αKG + Co)FIH-1, indicated a mixture of 5-coordinate and 6-coordinate enzyme forms, suggesting that resting FIH-1 can readily undergo uncoupled O₂-activation by loss of an H₂O ligand from the metal center.     

The increased expression of DEC1 gene is related to HIF-1α protein in gastric cancer cell lines

Overexpression of differentiated embryo chondrocyte 1 (DEC1) has been reported to contribute to the cellular differentiation, proliferation, and apoptosis of various cancers. Our previous studies have shown that DEC1 was highly expressed in gastric cancer (GCa) tissues. However, there is no report about the expression of DEC1 in GCa cell lines until now. In this study, We evaluated the mRNA and protein expression of DEC1 and hypoxia-inducible factor 1α (HIF-1α) under normoxic and hypoxic conditions in six GCa cell lines: BGC-823, MGC80-3, MKN1, AGS, FU97 and SGC-7901. An HIF-1α protein inhibitor was used to analyze the association of DEC1 and HIF-1α expression. Under normoxia, the mRNA expression of both HIF-1α and DEC1 was moderate, whereas the protein expression of DEC1 was higher than that of HIF-1α. Hypoxia induced the mRNA expression of DEC1 and the protein expression of HIF-1α and DEC1 in a time-dependent manner but had no effect on the mRNA expression of HIF-1α. Furthermore, inhibition of HIF-1α protein expression resulted in a significant decrease in both the mRNA and protein expression of DEC1. Taken together, DEC1 expression is correlated with HIF-1α protein in GCa cell line, blockage of HIF-1α protein led to reduced DEC1 expression. The efficacy of inhibiting HIF-1α and DEC1 expression should be tested in clinical trials as possible treatment for GCa.     

Differences in hydroxylation and binding of Notch and HIF-1α demonstrate substrate selectivity for factor inhibiting HIF-1 (FIH-1)

FIH-1, factor inhibiting hypoxia-inducible factor-1 (HIF-1), regulates oxygen sensing by hydroxylating an asparagine within HIF-α. It also hydroxylates asparagines in many proteins containing ankyrin repeats, including Notch1–3, p105 and IκBα. Relative binding affinity and hydroxylation rate are crucial determinants of substrate selection and modification. We determined the contributions of substrate sequence composition and length and of oxygen concentration to the FIH-1-binding and/or hydroxylation of Notch1–4 and compared them with those for HIF-1α. We also demonstrated hydroxylation of two asparagines in Notch2 and 3, corresponding to Sites 1 and 2 of Notch1, by mass spectrometry for the first time.Our data demonstrate that substrate length has a much greater influence on FIH-1-dependent hydroxylation of Notch than of HIF-1α, predominantly through binding affinity rather than maximal reaction velocity. The K_m value of FIH-1 for Notch1, <0.2 μM, is at least 250-fold lower than that of 50 μM for HIF-1α. Site 1 of Notch1–3 appeared the preferred site of FIH-1 hydroxylation in these substrates. Interestingly, binding of Notch4 to FIH-1 was observed with an affinity almost 10-fold lower than for Notch1–3, but no hydroxylation was detected. Importantly, we demonstrate that the K_m of FIH-1 for oxygen at the preferred Site 1 of Notch1–3, 10–19 μM, is an order of magnitude lower than that for Site 2 or HIF-1α. Hence, at least during in vitro hydroxylation, Notch is likely to become efficiently hydroxylated by FIH-1 even under relatively severe hypoxic conditions, where HIF-1α hydroxylation would be reduced.     

Effect of hypoxia-inducible factor-1alpha silencing on the sensitivity of human brain glioma cells to doxorubicin and etoposide

Multidrug resistance (MDR) is a significant problem underlying the poor prognosis associated with gliomas. Hypoxia-inducible factor-1α (HIF-1α) is thought to induce the genes expression involved in MDR. To evaluate the effect of silencing HIF-1α in human glioma T98G cells, cells were transfected with HIF-1α-small interference RNA (HIF-1α-siRNA) and cultured under hypoxic conditions. The effect of HIF-1α-siRNA on HIF-1α and multidrug resistance-associated protein 1 gene (MRP1) and protein levels was determined. Silencing rates of HIF-1α were 90%, 85%, and 88% at 24, 48, 72 h post-transfection, respectively. Corresponding rates of HIF-1α protein were 74.5%, 61.1% and 59.1%. MRP1 protein levels decreased by 7.6%, 36.8% and 45.2%. HIF-1α-siRNA transfected cells were significantly more sensitive to doxorubicin and etoposide compared to non-transfected cells. These findings suggest that the HIF-1α plays a role in mediating chemotherapeutic drug resistance in glioma cells. HIF-1α silencing may prove to be an effective therapeutic means of treating gliomas.     

Hesperidin inhibits expression of hypoxia inducible factor-1 alpha and inflammatory cytokine production from mast cells

The citrus unshiu peel has been used traditionally as a medicine to improve bronchial and asthmatic conditions or cardiac and blood circulation in Korea, China, and Japan. Here, we report the effects of citrus unshiu peel water extract (CPWE) on the phorbol myristate acetate (PMA) + calcium ionophore A23187-induced hypoxia-inducible factor-1α (HIF-1α) activation and inflammatory cytokine production from the human mast cell line, HMC-1 cells. We compared CPWE with hesperidin, a common constituent of citrus unshiu. CPWE and hesperidin inhibited the PMA + A23187-induced HIF-1α expression and the subsequent production of vascular endothelial growth factor (VEGF). In addition, CPWE suppressed PMA + A23187-induced phosphorylation of the extracellular signal-regulated kinase (ERK). We also show that the increased cytokines interleukin (IL)-1β, IL-8, and tumor necrosis factor (TNF)-α level was significantly inhibited by treatment of CPWE or hesperidin. In the present study, we report that CPWE and hesperidin are inhibitors of HIF-1α and cytokines on the mast cell-mediated inflammatory responses.     

HIF-1α and iNOS levels in crucian carp gills during hypoxia-induced transformation

Hypoxia inducible factor 1 alpha (HIF-1α) initiates expression of a wide variety of genes, some of which are involved in apoptosis and cell cycle arrest. We have previously shown that crucian carp increases its respiratory surface area 7.5-fold in response to hypoxia. This change is due to apoptosis and cell cycle arrest in specific parts of its gills. Here we have characterized crucian carp HIF-1α, and measured mRNA, protein and DNA binding levels during hypoxia exposure in crucian carp gills. We have also measured an HIF-1α-induced gene, the inducible nitric oxide synthase (iNOS), which has the ability to initiate apoptosis and cell cycle arrest. Crucian carp HIF-1α was found to have all critical domains known to be important for function. Comparison of the peptide sequence with other species indicated high similarity with other cyprinid fish, but a pronounced variation compared to the salmonid, rainbow trout. Further, we found HIF-1α protein to be stabilized during hypoxia. Further, HIF-1α was often present in normoxia, and showed marked individual weight-dependent variation. We found no alteration of iNOS mRNA levels during hypoxia exposure. These findings suggest HIF-1α involvement in hypoxia-induced change of respiratory surface area in crucian carp gills. However, its activity does not seem to be mediated through iNOS.     

Hypoxia-Inducible Factor 1 Regulation through Cross Talk between mTOR and MT1-MMP

Hypoxia-inducible factor 1 (HIF-1) plays a key role in the cellular adaptation to hypoxia. Although HIF-1 is usually strongly suppressed by posttranslational mechanisms during normoxia, HIF-1 is active and enhances tumorigenicity in malignant tumor cells that express the membrane protease MT1-MMP. The cytoplasmic tail of MT1-MMP, which can bind a HIF-1 suppressor protein called factor inhibiting HIF-1 (FIH-1), promotes inhibition of FIH-1 by Mint3 during normoxia. To explore possible links between HIF-1 activation by MT1-MMP/Mint3 and tumor growth signals, we surveyed a panel of 252 signaling inhibitors. The mTOR inhibitor rapamycin was identified as a possible modulator, and it inhibited the mTOR-dependent phosphorylation of Mint3 that is required for FIH-1 inhibition. A mutant Mint3 protein that cannot be phosphorylated exhibited a reduced ability to inhibit FIH-1 and promoted tumor formation in mice. These data suggest a novel molecular link between the important hub proteins MT1-MMP and mTOR that contributes to tumor malignancy.     

Evidence that two enzyme-derived histidine ligands are sufficient for iron binding and catalysis by factor inhibiting HIF (FIH)

A 2-His-1-carboxylate triad of iron binding residues is present in many non-heme iron oxygenases including the Fe(II) and 2-oxoglutarate (2OG)-dependent dioxygenases. Three variants (D201A, D201E, and D201G) of the iron binding Asp-201 residue of an asparaginyl hydroxylase, factor inhibiting HIF (FIH), were made and analyzed. FIH-D201A and FIH-D201E did not catalyze asparaginyl hydroxylation, but in the presence of a reducing agent, they displayed enhanced 2OG turnover when compared with wild-type FIH. Turnover of 2OG by FIH-D201A was significantly stimulated by the addition of HIF-1alpha(786-826) peptide. Like FIH-D201A and D201E, the D201G variant enhanced 2OG turnover but rather unexpectedly catalyzed asparaginyl hydroxylation. Crystal structures of the FIH-D201A and D201G variants in complex with Fe(II)/Zn(II), 2OG, and HIF-1alpha(786-826/788-806) implied that only two FIH-based residues (His-199 and His-279) are required for metal binding. The results indicate that variation of 2OG-dependent dioxygenase iron-ligating residues as a means of functional assignment should be treated with caution. The results are of mechanistic interest in the light of recent biochemical and structural analyses of non-heme iron and 2OG-dependent halogenases that are similar to the FIH-D201A/G variants in that they use only two His-residues to ligate iron.     

HIF-1α-dependent gene expression program during the nucleic acid-triggered antiviral innate immune responses

Recent studies suggest a novel role of HIF-1α under non-hypoxic conditions, including antibacterial and antiviral innate immune responses. However, the identity of the pathogen-associated molecular pattern which triggers HIF-1α activation during the antiviral response remains to be identified. Here, we demonstrate that cellular administration of double-stranded nucleic acids, the molecular mimics of viral genomes, results in the induction of HIF-1α protein level as well as the increase in HIF-1α target gene expression. Whole-genome DNA microarray analysis revealed that double-stranded nucleic acid treatment triggers induction of a number of hypoxia-inducible genes, and induction of these genes are compromised upon siRNA-mediated HIF-1α knock-down. Interestingly, HIF-1α knock-down also resulted in down-regulation of a number of genes involved in antiviral innate immune responses. Our study demonstrates that HIF-1α activation upon nucleic acid-triggered antiviral innate immune responses plays an important role in regulation of genes involved in not only hypoxic response, but also immune response. These authors contributed equally to this work.     

Hypoxia-mimetic agents desferrioxamine and cobalt chloride induce leukemic cell apoptosis through different hypoxia-inducible factor-1α independent mechanisms

Hypoxia presents pro-apoptotic and anti-apoptotic biphasic effects that appear to be dependent upon cell types and conditions around cells. The substantial reports demonstrated that commonly used hypoxia-mimetic agents cobalt chloride (CoCl₂) and desferrioxamine (DFO) could also induce apoptosis in many different kinds of cells, but the mechanism was poorly understood. In this work, we compare the apoptosis-inducing effects of these two hypoxia-mimetic agents with acute myeloid leukemic cell lines NB4 and U937 as in vitro models. The results show that both of them induce these leukemic cells to undergo apoptosis with a loss of mitochondrial transmembrane potentials (ΔΨ m), the activation of caspase-3/8 and the cleavage of anti-apoptotic protein Mcl-1, together with the accumulation of hypoxia-inducible factor-1 alpha (HIF-1α) protein, a critical regulator for the cellular response to hypoxia. Metavanadate and sodium nitroprusside significantly abrogate DFO rather than CoCl₂-induced mitochondrial Δ Ψ m collapse, caspase-3/8 activation, Mcl-1 cleavage and apoptosis, but they fail to influence DFO and CoCl₂-induced HIF-1α protein accumulation. Moreover, inducible expression of HIF-1α gene dose not alter DFO and CoCl₂-induced apoptosis in U937 cells. In conclusion, these results propose that although both DFO and CoCl₂-induced leukemic cell apoptosis by mitochondrial pathway-dependent and HIF-1α-independent mechanisms, DFO and CoCl₂-induced apoptosis involves different initiating signal pathways that remain to be investigated.     

HIF-1-Dependent Induction of Jumonji Domain-Containing Protein (JMJD) 3 under Hypoxic Conditions

Jumonji domain-containing proteins (JMJD) catalyze the oxidative demethylation of a methylated lysine residue of histones by using O₂, α-ketoglutarate, vitamin C, and Fe(II). Several JMJDs are induced by hypoxic stress to compensate their presumed reduction in catalytic activity under hypoxia. In this study, we showed that an H3K27me3 specific histone demethylase, JMJD3 was induced by hypoxia-inducible factor (HIF)-1α/β under hypoxia and that treatment with Clioquinol, a HIF-1α activator, increased JMJD3 expression even under normoxia. Chromatin immunoprecipitation (ChIP) analyses showed that both HIF-1α and its dimerization partner HIF-1β/Arnt occupied the first intron region of the mouse JMJD3 gene, whereas the HIF-1α/β heterodimer bound to the upstream region of the human JMJD3, indicating that human and mouse JMJD3 have hypoxia-responsive regulatory regions in different locations. This study shows that both mouse and human JMJD3 are induced by HIF-1.