Hypoxia-inducible Factor-1 Binding Research Articles

Hypoxia induces heat shock protein HSP70-2 expression in a HIF-1 dependent manner

To investigate role of hypoxia inducible factor 1 (HIF-1) in the transcriptional activation of heat shock protein 70-2 (HSP70-2) in hepatocellular carcinoma (HCC) cells under hypoxic conditions. HCC cells were exposed to reduced oxygen atmosphere (1% O2), or treated with YC-1 or HIF-1 alpha siRNA, the expression of HIF-1 alpha and HSP70-2 were detected by Western blot analysis. Serial deletions of the HSPA2 promoter were cloned in the reporter pGL3-Basic plasmid. These reporter plasmids were co-transfected with HIF-1 alpha siRNA, and the promoter activities were detected with the dual luciferase assay. Western blot analysis showed that both HIF-1 alpha and HSP70-2 proteins were strongly increased after HCC cells were exposed to hypoxic conditions (1% O2) for 6 h, and the expression level of HSP70-2 was increased in a time-dependent manner. Treatment of HepG2 cells with YC-1 or HIF-1 alpha siRNA significantly inhibited the expression of HIF-1 alpha and HSP70-2. In silico analysis of the HSP70-2 promoter using the Gene2 Promoter software revealed the presence of two putative hypoxic response element (HRE) consensus at -446bp (HRE1) and -238bp (HRE2). Depletion of promoter sequence between -653 and -385 led to a dramatic reduction of promoter activity, whereas further deletion to position -201 did not reduce the activity further. These data suggested that HRE1 plays an important role in hypoxia-induced activation of the HSPA2 promoter. Site-directed mutagenesis further confirmed these results. Mutation of HRE1 but not of HRE2 abrogated the sensitivity of the HSP70-2 promoter to hypoxia. HSP70-2 expression is up-regulated in response to hypoxia and a HIF-1 binding site (HRE1) in the HSP70-2 promoter is involved in this response.

The redox state of glutathione regulates the hypoxic induction of HIF-1

Hypoxia inducible factor 1 (HIF-1) regulates the transcription of vascular endothelial growth factor (VEGF), which plays important roles in angiogenesis. We investigated the redox effect of glutathione (GSH) on the hypoxic induction of HIF-1 in a human oral squamous cell carcinoma (HSC-2) cell line. The maximal induction of HIF-1 in HSC-2 cells was observed 30 h after hypoxia, and VEGF mRNA was expressed after 36 h under hypoxia. GSH ethyl ester (GSHee, a membrane permeable analog of GSH) and N-acetyl-L-cysteine (NAC, a membrane permeable precursor of GSH) reduced HIF-1 binding activity in a dose-dependent manner. Further, HIF-1 dependent promoter activity was similarly reduced by GSHee and NAC. However, ebselen, which increases glutathione peroxidase activity and oxidizes GSH, negated the effect of GSHee on HIF-1 dependent promoter activity. The inhibitory effect of GSHee and NAC on HIF-1 binding activity was reversed by bis (2-chlorethyl)-nitrosourea, an oxidized glutathione (GSSG) reductase inhibitor which increases the concentration of GSSG. GSSG methyl ester (GSSGme), a membrane permeable analog of GSSG, enhanced HIF-1 dependent promoter activity and exhibited a bell-shaped concentration-dependant activity curve. The increasing effect of GSSGme on HIF-1 induction was also observed under chemically-induced hypoxia obtained using cobalt chloride. These results suggest that changes in the intracellular GSSG/GSH ratio may regulate HIF-1 induction during hypoxia.

Hypoxia‐mediated up‐regulation of MGr1‐Ag/37LRP in gastric cancers occurs via hypoxia‐inducible‐factor 1‐dependent mechanism and contributes to drug resistance

Our previous study demonstrated hypoxia-inducible factor-1(HIF-1) could prompt multidrug resistance (MDR) phenotype and MGr1-Ag/37LRP, a novel drug-resistance protein was reported by our labortary, associated with multidrug resistance in gastric cancer. Given this association, we hypothesized that MGr1-Ag/37LRP contributed to HIF-1-dependent hypoxia-induced MDR phenotype. Initial experiments revealed that blocking MGr1-Ag/37LRP expression by siRNA in gastric cancer cells effectively reversed multidrug resistance phenotype induced by hypoxia. Subsequent analysis of MGr1-Ag/37LRP mRNA and protein in gastric cancer cells revealed a time-dependent manner increase with hypoxia. While the up-regulation of MGr1-Ag/37LRP was abolished by HIF-1 inhibition with siRNA. Studies using luciferase promoter constructs revealed a significant increase in activity in cells subject to hypoxia and such hypoxia inducibility was lost in cells co-transfected siRNA targeting HIF-1. Analysis of the MGr1-Ag/37LRP promoter revealed several potential binding sites for HIF-1. Electrophoretic mobility shift assay and chromatin immunoprecipitation demonstrated a functional HIF-1 binding site within MGr1-Ag/37LRP gene regulatory sequence located at -16 to -11 relative to the transcriptional initiation point. These observations demonstrate that MGr1-Ag/37LRP is actively engaged by hypoxia and represent a novel HIF-1 target. Such results suggest hypoxia-elicited MGr1-Ag/37LRP expression as a pathway for resistance of gastric cancer to chemotherapeutics.

Preferential binding of HIF-1 to transcriptionally active loci determines cell-type specific response to hypoxia

ChIP-chip and microarray expression studies show that, in response to hypoxia, HIF-1 preferentially binds to and up-regulates already active genes.

An Algorithm for Identifying Novel Targets of Transcription Factor Families: Application to Hypoxia-inducible Factor 1 Targets

Efficient and effective analysis of the growing genomic databases requires the development of adequate computational tools. We introduce a fast method based on the suffix tree data structure for predicting novel targets of hypoxia-inducible factor 1 (HIF-1) from huge genome databases. The suffix tree data structure has two powerful applications here: one is to extract unknown patterns from multiple strings/sequences in linear time; the other is to search multiple strings/sequences using multiple patterns in linear time. Using 15 known HIF-1 target gene sequences as a training set, we extracted 105 common patterns that all occur in the 15 training genes using suffix trees. Using these 105 common patterns along with known subsequences surrounding HIF-1 binding sites from the literature, the algorithm searches a genome database that contains 2,078,786 DNA sequences. It reported 258 potentially novel HIF-1 targets including 25 known HIF-1 targets. Based on microarray studies from the literature, 17 putative genes were confirmed to be upregulated by HIF-1 or hypoxia inside these 258 genes. We further studied one of the potential targets, COX-2, in the biological lab; and showed that it was a biologically relevant HIF-1 target. These results demonstrate that our methodology is an effective computational approach for identifying novel HIF-1 targets.

Selective induction of integrin βi by hypoxia‐inducible factor: implications for wound healing

Because of localized vascular damage and increased tissue oxygen demand, wound healing occurs in a relatively hypoxic microenvironment. These features are particularly relevant to wound healing and fibrosis in chronic inflammatory conditions, such as Crohn's disease and ulcerative colitis. In these studies, we sought to identify the contribution of hypoxia to mechanisms of wound repair in a model of the intestinal submucosa. Initial studies revealed that hypoxia promotes wound healing, as modeled by an increase in intestinal fibroblast-mediated collagen gel contraction. Guided by results from transcriptional profiling, we identified the selective induction of fibroblast integrin beta1 (ITGB1) by hypoxia. Further analysis revealed that hypoxia, as well as pharmacological activators of hypoxia-inducible factor (HIF), induce fibroblast beta1 integrin mRNA, protein, and function by as much as 4-fold. Cloning and analysis of the beta1 integrin gene promoter revealed a 10 +/- 0.8-fold increase in promoter activity in response to hypoxia, and subsequent studies identified a functional DNA binding region for HIF in the ITGB1 gene promoter. Mutational analysis of the HIF binding site within the ITGB1 promoter resulted in a significant loss of ITGB1 hypoxia-inducibility. As proof of principle, studies in a murine model of colitis revealed a correlation between colitic disease severity and tissue ITGB1 expression (R(2)=0.80). Taken together, these results demonstrate that hypoxia induces fibroblast ITGB1 expression and function by transcriptional mechanisms dependent on HIF.

Transcriptional up‐regulation of FoxM1 in response to hypoxia is mediated by HIF‐1

The proliferation-specific Forkhead box M1 (FoxM1) transcription factor is overexpressed in cancer cells and acts as an important regulator of cancer cell growth and survival. Here, we show the molecular mechanisms by which hypoxia regulate FoxM1 expression in cancer cells. When cells were subjected to hypoxia (1% O2), the mRNA and protein levels of FoxM1 had a significant increase in cancer cells (HepG2, MCF-7, and HeLa). Such increase was due to the direct binding of hypoxia-inducible factor 1 (HIF-1) to the HIF-1 binding sites in the FoxM1 promoter. By deletion and mutation assays, we demonstrated that the HIF1-1 and HIF1-3/4 binding sites on the FoxM1 promoter were essential for transcriptional activation of FoxM1 by hypoxia. We also demonstrated that HIF-1alpha directly bound to the promoter of FoxM1 and the binding was specific, as revealed by HIF-1 binding/competition assay and chromatin immunoprecipitation assay. Consequently, the up-regulation of FoxM1 accelerated the growth of hypoxic cancer cells by decreasing nuclear levels of p21 and increasing expression of cyclin B1 and cyclin D1. These findings provide a new insight into how tumor cells overcome hypoxic stress and survive, and also disclose a new regulatory mechanism of FoxM1 expression in tumor cells.

Impaired Function of Hematopoietic Stem Cells with Abrogated Hypoxia Induced Expression of Vascular Endothelial Growth Factor

Hypoxia has been suggested to be a characteristic of the bone marrow (BM) microenvironment where hematopoietic stem cells (HSC) reside. The major molecular response to hypoxia is the activation of hypoxia inducible factors (HIF). These transcription factors activate gene expression by binding to hypoxia response elements (HRE) in the promoter region of target genes, such as vascular endothelial growth factor (VEGF). It has been demonstrated that VEGF is indispensable for HSC survival in the adult and that VEGF mediated HSC survival is cell intrinsic. We are therefore investigating the role of hypoxia-regulated VEGF expression in HSC survival. For our studies we use the transgenic mouse model VEGFδ/δ. In this model, the HRE in the VEGF promoter is mutated which prevents HIF binding. Thus, hypoxia-induced VEGF transcriptional activation is abrogated while basal VEGF expression remains unaffected. Steady state hematopoiesis was not affected by the mutation since distribution between the B-cell, T-cell and myeloid lineages in both peripheral blood and BM of mice homozygous for the mutation was comparable to wild type animals. Furthermore, we could not detect any differences in the frequency of lineage-, Sca1+, c-kit+, CD34- cells in the BM between VEGFδ/δ and wild type mice. However, when assessing HSC functional properties in transplantation assays, BM from mice homozygous for the mutation was demonstrated to have dramatically reduced HSC function. Only around 60 % of lethally irradiated recipients that were transplanted with VEGFδ/δ BM could be rescued while recipients receiving wild type BM survived as expected. In addition, during early hematopoietic reconstitution, a skewing towards myeloid differentiation at the expense of B-cell differentiation could be observed in peripheral blood of recipients reconstituted with VEGFδ/δ BM. Moreover, in a competitive repopulation assay, VEGFδ/δ BM was shown to have a severe competitive disadvantage compared to wild type BM. Our findings indicate an important role for hypoxia related VEGF expression in reconstituting HSCs and suggest that low oxygen levels might be an important characteristic of the HSC niche.

Transcriptional upregulation of HSP70‐2 by HIF‐1 in cancer cells in response to hypoxia

Heat shock protein 70-2 (HSP70-2) can be expressed by cancer cells and act as an important regulator of cancer cell growth and survival. Here, we show the molecular mechanisms by which hypoxia regulate HSP70-2 expression in cancer cells. When cells were subjected to hypoxia (1% O2), the expression of HSP70-2 had a significant increase in cancer cells. Such increase was due to the direct binding of hypoxia-inducible factor to hypoxia-responsive elements (HREs) in the HSP70-2 promoter. By luciferase assays, we demonstrated that the HRE1 at position -446 was essential for transcriptional activation of HSP70-2 promoter under hypoxic conditions. We also demonstrated that HIF-1alpha binds to the HSP70-2 promoter and the binding is specific, as revealed by HIF binding/competition and chromatin immunoprecipitation assays. Consequently, the upregulation of HSP70-2 enhanced the resistance of tumor cells to hypoxia-induced apoptosis. These findings provide a new insight into how tumor cells overcome hypoxic stress and survive, and also disclose a new regulatory mechanism of HSP70-2 expression in tumor cells.

Induction of Pyruvate Dehydrogenase Kinase-3 by Hypoxia-inducible Factor-1 Promotes Metabolic Switch and Drug Resistance

The switch of cellular metabolism from mitochondrial respiration to glycolysis is the hallmark of cancer cells and associated with tumor malignancy. However, the mechanism of this metabolic switch remains largely unknown. Herein, we reported that hypoxia-inducible factor-1 (HIF-1) induced pyruvate dehydrogenase kinase-3 (PDK3) expression leading to inhibition of mitochondrial respiration. Promoter activity assay, small interference RNA knockdown assay, and chromatin immunoprecipitation assay demonstrated that hypoxia-induced PDK3 gene activity was regulated by HIF-1 at the transcriptional level. Forced expression of PDK3 in cancer cells resulted in increased lactic acid accumulation and drugs resistance, whereas knocking down PDK3 inhibited hypoxia-induced cytoplasmic glycolysis and cell survival. These data demonstrated that increased PDK3 expression due to elevated HIF-1alpha in cancer cells may play critical roles in metabolic switch during cancer progression and chemoresistance in cancer therapy.

Enhancement of Angiogenesis Through Stabilization of Hypoxia-inducible Factor-1 by Silencing Prolyl Hydroxylase Domain-2 Gene

Hypoxia-inducible factor-1 (HIF-1) plays a central role in cellular response to hypoxia by activating vascular endothelial growth factor (VEGF) and other angiogenic factors. Prolyl hydroxylase domain-2 (PHD2) protein induces the degradation of HIF-1 by hydroxylating specific prolyl residues. Therefore gene silencing of PHD2 by RNA interference (RNAi) might increase the expression of angiogenic growth factors and, consequently, neoangiogenesis through the stabilization of HIF-1alpha. In this study we have shown that the specific silencing of PHD2 is sufficient for stabilizing HIF-1alpha and increasing its transcriptional activity, resulting in the increased expression of angiogenic factors including VEGF and fibroblast growth factor-2 (FGF2). Moreover, when PHD2-siRNA vector was used, the increase in VEGF secretion was observed for as long as 18 days after transfection. In vitro treatment of human umbilical vein endothelical cells with conditioned medium from PHD2-siRNA vector-transfected NIH3T3 cells was shown to increase cell proliferation. Also, in vivo angiogenesis was observed in mice implanted with Matrigel plugs mixed with NIH3T3 cells transfected with PHD2-siRNA vector. These results indicate that PHD2 silencing induces expressions of multiple angiogenic growth factors by stabilizing HIF-1alpha, and that the implantation of cells transfected with PHD2-siRNA vector is sufficient to enhance angiogenesis in vivo. In the light of these findings, PHD2 silencing by RNAi might offer a potential tool for angiogenic therapy.

Hypoxia upregulates the histone demethylase JMJD1A via HIF-1

The histone demethylase Jumonji domain containing 1A (JMJD1A) demethylates H3K9 residues and thereby transactivates distinct target genes. Investigating the effect of hypoxia on JMJD1A expression, we found increased JMJD1A mRNA in different organs of rats exposed to normobaric hypoxia (8% O 2). Compared to adult samples, JMJD1A was increased in most tissues of human fetuses in whom oxygen supply is low compared to postnatal levels. Upregulation of JMJD1A mRNA and protein in cultured human cells exposed to hypoxia or iron scavengers in vitro was abrogated when hypoxia-inducible factor-1 (HIF-1) signaling was blocked by siRNAs. A single pivotal hypoxia responsive element (HRE) in the promoter of the human JMJD1A gene was identified that mediates JMJD1A upregulation by hypoxia, iron scavengers, and HIF-1. These findings demonstrate that JMJD1A can be stimulated by hypoxia both in vitro and in vivo involving binding of HIF-1 to a specific HRE in the JMJD1A promoter.

Heterogeneity in binding and gene-expression regulation by HIF-2α

Here, we demonstrate by chromatin immunoprecipitation that the binding of hypoxia-inducible factors to gene regulatory regions is differentially influenced in cancer cells. Binding of HIF-2α varies depending on hypoxic conditions, although HIF-1α is constantly bound to these regions. We found by RNA interference experiments that HIF-2α plays a minor role in VEGF gene upregulation under hypoxia or CoCl 2 treatment, even when both HIFs are similarly bound to the promoter region. HIF-2α activated or suppressed the ENO1 gene under various conditions, irrespective of promoter binding. We additionally found that HIF dependence on EPO gene induction could be altered depending on the conditions, irrespective of the binding pattern of HIFs. These results demonstrate that, unlike HIF-1α, HIF-2α differentially binds and regulates transcription under hypoxia.

Rational design of minimal hypoxia-inducible enhancers

The hypoxia-inducible factor (HIF) activates transcription via binding to the highly variable hypoxia-responsive elements (HREs). All hypoxia-inducible constructs described to date utilize multimers of naturally occurring HREs. Here, we describe the rational design of minimal hypoxia-inducible enhancers, conceptually equivalent to using an optimized HIF-binding site (HBS) as the building block. Optimizations of the HBS, spacing between HBSs, the distance from the minimal promoter, and orientation of HBSs allowed us to design constructs with high hypoxic activity. Activation of the 4xopt HBS (36 bp) construct by hypoxia or HIF-1α and HIF-2α was comparable with that of the 4x EPO HRE (208 bp) construct. The strong synergism between the properly arranged optimized HBSs was due to stimulation of high affinity HIF binding. Our data prove, for the first time, that it is possible to assemble artificial hypoxia-inducible enhancers from a single type of regulatory element-optimized HBS.

Transglutaminase 2 protects against ischemic insult, interacts with HIF1β, and attenuates HIF1 signaling

Transglutaminase 2 (TG2) is a multifunctional enzyme that has been implicated in the pathogenesis of neurodegenerative diseases, ischemia, and stroke. The mechanism by which TG2 modulates disease progression have not been elucidated. In this study we investigate the role of TG2 in the cellular response to ischemia and hypoxia. TG2 is up-regulated in neurons exposed to oxygen and glucose deprivation (OGD), and increased TG2 expression protects neurons against OGD-induced cell death independent of its transamidating activity. We identified hypoxia inducible factor 1beta (HIF1beta) as a TG2 binding partner. HIF1beta and HIF1alpha together form the heterodimeric transcription factor hypoxia inducible factor 1 (HIF1). TG2 and the transaminase-inactive mutant C277S-TG2 inhibited a HIF-dependent transcription reporter assay under hypoxic conditions without affecting nuclear protein levels for HIF1alpha or HIF1beta, their ability to form the HIF1 heterodimeric transcription factor, or HIF1 binding to its DNA response element. Interestingly, TG2 attenuates the up-regulation of the HIF-dependent proapoptotic gene Bnip3 in response to OGD but had no effect on the expression of VEGF, which has been linked to prosurvival processes. This study demonstrates for the first time that TG2 protects against OGD, interacts with HIF1beta, and attenuates the HIF1 hypoxic response pathway. These results indicate that TG2 may play an important role in protecting against the delayed neuronal cell death in ischemia and stroke.

A possible role for hypoxia-induced apelin expression in enteric cell proliferation

Apelin is the endogenous ligand for the APJ receptor, and apelin and APJ are expressed in the gastrointestinal (GI) tract. Intestinal inflammation increases intestinal hypoxia-inducible factor (HIF) and apelin expression. Hypoxia and inflammation are closely linked cellular insults. The purpose of these studies was to investigate the influence of hypoxia on enteric apelin expression. Exposure of rat pups to acute hypoxia increased hepatic, stomach-duodenal, and colonic apelin mRNA levels 10-, 2-, and 2-fold, respectively (P < 0.05 vs. controls). Hypoxia also increased colonic APJ mRNA levels, and apelin treatment during hypoxia exposure enhanced colonic APJ mRNA levels further. In vitro hypoxia also increased apelin and APJ mRNA levels. The hypoxia-induced elevation in apelin expression is most likely mediated by HIF, since HIF-activated apelin transcriptional activity is dependent on an intact, putative HIF binding site in the rat apelin promoter. Acute exposure of rat pups to hypoxia lowered gastric and colonic epithelial cell proliferation; hypoxia in combination with apelin treatment increased epithelial proliferation by 50%. In vitro apelin treatment of enteric cells exposed to hypoxia increased cell proliferation. Apelin treatment during normoxia was ineffective. Our studies imply that the elevation in apelin expression during hypoxia and inflammation in the GI tract functions in part to stimulate epithelial cell proliferation.

Allosteric effects in the marginally stable von Hippel–Lindau tumor suppressor protein and allostery-based rescue mutant design

Many multifunctional tumor suppressor proteins have low stability, a property linked to cancer development. The von Hippel-Lindau tumor suppressor protein (pVHL) is one of these proteins. pVHL forms part of the E3 ubiquitin ligase complex that regulates the degradation of the hypoxia-inducible factor (HIF). Under native conditions, free pVHL is a molten globule, but it is stabilized in the E3 complex. By using molecular dynamics simulations, we observed that the interface between the two pVHL domains is the least stable region in unbound pVHL. We designed five stable mutants: one with a mutation at the interdomain interface and the others in the alpha- or beta-domains. Experimentally, type 2B pVHL disease mutant Y98N at the HIF binding site was shown to destabilize pVHL and decrease its binding affinity to HIF. Our simulations showed that the decrease in pVHL stability and binding affinity are allosterically regulated. The mutations designed to stabilize unbound wild-type pVHL, which are away from the elongin C and HIF binding sites, successfully stabilized the Y98N pVHL-elongin C complex and lowered the binding free energy of pVHL with HIF. Our results indicated both the enthalpic and dynamic allosteric components between the elongin C and HIF binding sites in pVHL, in the alpha- and beta-domains, respectively, mediated by the interdomain interface and linker. Drugs mimicking the allosteric effects of these mutants may rescue pVHL function in von Hippel-Lindau disease.

Synergystic induction of HIF-1α transcriptional activity by hypoxia and lipopolysaccharide in macrophages

Hypoxia Inducible Factor-1 (HIF-1) is activated by a variety of stimuli, including inflammatory mediators. In this report we investigated the role that bacterial lipopolysaccharide (LPS) and hypoxia play in the regulation of HIF-1-dependent gene expression in macrophages. We report that murine macrophages stimulated with low concentrations of LPS (1-10 ng/ml) expressed significantly higher levels of inducible nitric oxide synthase (iNOS) mRNA when cultured under hypoxic compared to normoxic conditions. Functional studies of the iNOS promoter demonstrated that the synergistic interaction between LPS and hypoxia was mediated, at least in part, by the NFκB and the HIF-1 binding sites. In addition, transient transfection experiments using a Hypoxia Response Element (HRE)-containing plasmid showed that LPS and hypoxia synergistically induced HIF-1-dependent transcriptional activity. Interestingly, LPS did not significantly affect HIF-1α protein levels or HIF-1 DNA binding activity relative to hypoxic induction. HIF-1α, but not HIF-2α, was critical for the synergistic induction of HRE-dependent transcriptional activity in macrophages, as indicated by experiments using siRNA targeting HIF-1α or HIF-2α. Addition of ROS-scavengers completely abrogated the synergistic induction of HIF-1 transcriptional activity by LPS and hypoxia, but neither inhibited HIF-1 transcriptional activity induced by hypoxia alone nor affected HIF-1α protein levels or HIF-1 DNA binding induced by hypoxia alone or hypoxia plus LPS. Taken together, our results demonstrate that LPS and hypoxia act synergistically to induce HIF-1α-transcriptional activity and they emphasize the existence of a cross talk between hypoxic and non-hypoxic signaling pathways in the regulation of macrophages gene expression.

Hypoxia-inducible Factor-1 Mediates Neuronal Expression of the Receptor for Advanced Glycation End Products following Hypoxia/Ischemia

Activation of the receptor for advanced glycation endproducts (RAGE) by its multiple ligands can trigger diverse signaling pathways with injurious or pro-survival consequences. In this study, we show that Rage mRNA and protein levels were stimulated in the mouse brain after experimental stroke and systemic hypoxia. In both cases, RAGE expression was primarily associated with neurons. Activation of RAGE-dependent pathway(s) post-ischemia appears to have a neuroprotective role because mice genetically deficient for RAGE exhibited increased infarct size 24 h after injury. Up-regulation of RAGE expression was also observed in primary neurons subjected to hypoxia or oxygen-glucose deprivation, an in vitro model of ischemia. Treatment of neurons with low concentrations of S100B decreased neuronal death after oxygen-glucose deprivation, and this effect was abolished by a neutralizing antibody against RAGE. Conversely, high concentrations of exogenous S100B had a cytotoxic effect that seems to be RAGE-independent. As an important novel finding, we demonstrate that hypoxic stimulation of RAGE expression is mediated by the transcription factor hypoxia-inducible factor-1. This conclusion is supported by the finding that HIF-1alpha down-regulation by Cre-mediated excision drastically decreased RAGE induction by hypoxia or desferrioxamine. In addition, we showed that the mouse RAGE promoter region contains at least one functional HIF-1 binding site, located upstream of the proposed transcription start site. A luciferase reporter construct containing this RAGE promoter fragment was activated by hypoxia, and mutation at the potential HIF-1 binding site decreased hypoxia-dependent promoter activation. Specific binding of HIF-1 to this putative HRE in hypoxic cells was detected by chromatin immunoprecipitation assay.

Erythrocytosis Caused by Mutations in the PHD2 and VHL Genes.

Human diseases have provided important insights into the function of fundamental physiological processes, and studying erythrocytosis has increased our understanding of the oxygen sensing pathway. In some cases this rare disorder arises specifically from dysregulation of the erythropoietin (Epo) axis. Epo gene transcription is under the control of a negative feedback mechanism involving the kidneys, which sense tissue hypoxia at the molecular level via the hypoxia inducible factor (HIF) transcription complex. Both subunits of the HIF complex, alpha and beta, are constitutively expressed but only the beta subunit is detectable in the presence of oxygen. Key prolines in the oxygen degradation domain of HIFalpha are hydroxylated by a family of prolyl hydroxylase enzymes, PHD1-3. Of these PHD2 has the most widespread tissue distribution. Upon hydroxylation of HIF, the von Hippel Lindau (VHL) protein, a component of an E3 ligase complex, promotes the ubiquitination of the alpha subunit. HIFalpha is then targeted to the proteasome and the transcription of HIF target genes is prevented. In hypoxia the activity of the PHD enzymes is low and the HIF complex assembles causing upregulation of genes such as Epo. Over the last ten years we have maintained a registry of individuals with erythrocytosis. To date 181 patients have been included with clinical details and consented DNA samples have been collected. There is a preponderance of males with ratio of 1.7 males to each female. The mean age of erythrocytosis individuals on the registry is 37 years. The majority of erythrocytosis patients have inappropriately normal (46%) or raised (26%) serum Epo levels as compared to PV, where the Epo level is often undetectable. Thus it can be inferred that dysregulation of the Epo axis via the oxygen sensing pathway would be a significant cause of erythrocytosis. Consequently, PHD2 and VHL have been investigated. We have now identified 2 different mutations, Pro317Arg and Arg371His, in PHD2. In the tertiary structure of PHD2, Pro317and Arg371 are close to essential iron binding residues and furthermore suggest the location of a HIF binding groove. The Arg200Trp VHL mutation, commonly described as the Chuvash mutation, has been detected in 8 Asian families and their large kindred. Two Western European individuals were found to be compound heterozygous for the Arg200Trp and either the novel Pro192Thr or Gly144Arg variants. Intriguingly 2 further individuals possess one wild type and one mutated allele, Arg200Trp or Leu188Val. Screening other genes of the oxygen sensing pathway has failed to reveal further defects in these individuals. In summary, defects in the oxygen sensing pathway are associated with the development of erythrocytosis. Mutations in VHL are more common than PHD2 and highlight the importance of these proteins in the maintenance of red cell homeostasis.