Hypoxia-inducible Transcription Factor System Research Articles

Potent and Selective Triazole-Based Inhibitors of the Hypoxia-Inducible Factor Prolyl-Hydroxylases with Activity in the Murine Brain.

As part of the cellular adaptation to limiting oxygen availability in animals, the expression of a large set of genes is activated by the upregulation of the hypoxia-inducible transcription factors (HIFs). Therapeutic activation of the natural human hypoxic response can be achieved by the inhibition of the hypoxia sensors for the HIF system, i.e. the HIF prolyl-hydroxylases (PHDs). Here, we report studies on tricyclic triazole-containing compounds as potent and selective PHD inhibitors which compete with the 2-oxoglutarate co-substrate. One compound (IOX4) induces HIFα in cells and in wildtype mice with marked induction in the brain tissue, revealing that it is useful for studies aimed at validating the upregulation of HIF for treatment of cerebral diseases including stroke.

Acute hypoxia modifies regulation of neuroglobin in the neonatal mouse brain

Among endogenous adaptive systems to hypoxia, neuroglobin, a recently discovered heme protein, was suggested as a novel oxygen-dependent neuroprotectant. We aimed to characterize i) maturational age-related regulation of neuroglobin in the developing mouse brain under normoxic and hypoxic conditions, and ii) the role of hypoxia-inducible transcription factors (HIFs) as possible mediators of O2-dependent regulation of neuroglobin in vitro and in vivo.During early stages of postnatal brain maturation (P0–P14) neuroglobin mRNA levels significantly increased in developing mouse forebrains. By immunohistochemical analysis we confirmed expression of neuroglobin protein in the cytoplasm of developing neurons but not glial cells under normoxic conditions. Exposure of the immature brains (P0, P7) to acute (8% O2, 6h) and chronic systemic hypoxia (10% O2, 7days) led to differential activation of neuroglobin varying with maturational stage (P0, P7) and severity of hypoxia. This observation may indicate that neuroglobin is involved in adaptive responses of immature neurons to acute hypoxia during an early stage of mouse brain maturation (P0). In response to activation of the HIF system by prolyl-4-hydroxylase inhibitor (FG-4497), neuroglobin mRNA expression was significantly up-regulated in primary mouse cortical neurons (DIV6) exposed to normoxia and hypoxia (1% O2) compared to non-treated controls.In conclusion, present results strongly indicate that cerebral regulation of neuroglobin is related to maturational stage and that hypoxia-induced neuroglobin up-regulation is modified by the HIF system.

Hypoxia-Inducible Transcription Factors Stabilization in the Thick Ascending Limb Protects against Ischemic Acute Kidney Injury

Hypoxia-inducible transcription factors (HIF) protect cells against oxygen deprivation, and HIF stabilization before ischemia mitigates tissue injury. Because ischemic acute kidney injury (AKI) often involves the thick ascending limb (TAL), modulation of HIF in this segment may be protective. Here, we generated mice with targeted TAL deletion of the von Hippel-Lindau protein (Vhl), which mediates HIF degradation under normoxia, using Tamm-Horsfall protein (Thp)-driven Cre expression. These mice showed strong expression of HIF-1α in TALs but no changes in kidney morphology or function under control conditions. Deficiency of Vhl in the TAL markedly attenuated proximal tubular injury and preserved TAL function following ischemia-reperfusion, which may be partially a result of enhanced expression of glycolytic enzymes and lactate metabolism. These results highlight the importance of the thick ascending limb in the pathogenesis of AKI and suggest that pharmacologically targeting the HIF system may have potential to prevent and mitigate AKI.

Inhibition of Prolyl Hydroxylases Increases Erythropoietin Production in ESRD

The reasons for inadequate production of erythropoietin (EPO) in patients with ESRD are poorly understood. A better understanding of EPO regulation, namely oxygen-dependent hydroxylation of the hypoxia-inducible transcription factor (HIF), may enable targeted pharmacological intervention. Here, we tested the ability of fibrotic kidneys and extrarenal tissues to produce EPO. In this phase 1 study, we used an orally active prolyl-hydroxylase inhibitor, FG-2216, to stabilize HIF independent of oxygen availability in 12 hemodialysis (HD) patients, six of whom were anephric, and in six healthy volunteers. FG-2216 increased plasma EPO levels 30.8-fold in HD patients with kidneys, 14.5-fold in anephric HD patients, and 12.7-fold in healthy volunteers. These data demonstrate that pharmacologic manipulation of the HIF system can stimulate endogenous EPO production. Furthermore, the data indicate that deranged oxygen sensing--not a loss of EPO production capacity--causes renal anemia.

The hypoxia‐inducible transcription factor pathway regulates oxygen sensing in the simplest animal, Trichoplax adhaerens

The hypoxic response in humans is mediated by the hypoxia-inducible transcription factor (HIF), for which prolyl hydroxylases (PHDs) act as oxygen-sensing components. The evolutionary origins of the HIF system have been previously unclear. We demonstrate a functional HIF system in the simplest animal, Trichoplax adhaerens: HIF targets in T. adhaerens include glycolytic and metabolic enzymes, suggesting a role for HIF in the adaptation of basal multicellular animals to fluctuating oxygen levels. Characterization of the T. adhaerens PHDs and cross-species complementation assays reveal a conserved oxygen-sensing mechanism. Cross-genomic analyses rationalize the relative importance of HIF system components, and imply that the HIF system is likely to be present in all animals, but is unique to this kingdom.

Late-gestational systemic hypoxia leads to a similar early gene response in mouse placenta and developing brain

Late-gestational intrauterine hypoxia represents a well-known risk factor of acquired perinatal brain injury. Cell type and age-specific sensitivity of hypoxia-responsive genes to low-oxygen partial pressure is to be considered in the screening for early indicators of fetoplacental tissue hypoxia. To identify early hypoxia-induced alterations in gene expression during late-gestational hypoxia (6% O(2), 6 h; gestational day 20) we compared primary mouse placenta and brain transcriptomes using high-density oligonucleotide microarrays. Upregulation of candidate marker genes for hypoxia was confirmed by quantitative RT-PCR and immunohistochemistry. Both developing brain and placenta were highly responsive to systemic hypoxia at the level of gene expression involving hypoxia-inducible transcription factor (HIF)-dependent genes and immediate early genes (IEG) (Fos, Jun, Egr1, Bhlhb2), apoptosis-promoting factors (Bnip3, Dusp1, Ier3) that were all upregulated, and genes modulating RNA binding and translation (Rbm3, Thap2, Lig4, Rbm12b) that mainly were downregulated. Functional activity of the HIF system was obvious from elevated expression of various known HIF target genes (Adm, Vegf, Hk2, Pdk1, Bnip3, Ier3, Dusp-1), indicating immediate availability among early response to acute hypoxia. In addition, genes not yet described as being hypoxia related were identified that are involved in angiogenesis/cell differentiation (Gna13, Gab2), mRNA processing, and embryonic development. RT-PCR of placenta and brain tissues confirmed upregulation of selected HIF target genes and IEG. These data indicate that the early hypoxia-induced genomic response of the placenta mirrors that of developing brain in a temporally parallel manner. Our observations implicate future diagnostic options to identify fetal and cerebral tissue hypoxia.

Epidithiodiketopiperazines Block the Interaction between Hypoxia-inducible Factor-1α (HIF-1α) and p300 by a Zinc Ejection Mechanism

The hypoxic response in humans is regulated by the hypoxia-inducible transcription factor system; inhibition of hypoxia-inducible factor (HIF) activity has potential for the treatment of cancer. Chetomin, a member of the epidithiodiketopiperazine (ETP) family of natural products, inhibits the interaction between HIF-alpha and the transcriptional coactivator p300. Structure-activity studies employing both natural and synthetic ETP derivatives reveal that only the structurally unique ETP core is required and sufficient to block the interaction of HIF-1alpha and p300. In support of both cell-based and animal work showing that the cytotoxic effect of ETPs is reduced by the addition of Zn(2+) through an unknown mechanism, our mechanistic studies reveal that ETPs react with p300, causing zinc ion ejection. Cell studies with both natural and synthetic ETPs demonstrated a decrease in vascular endothelial growth factor and antiproliferative effects that were abrogated by zinc supplementation. The results have implications for the design of selective ETPs and for the interaction of ETPs with other zinc ion-binding protein targets involved in gene expression.

Hydroxylation of Hypoxia-Inducible Transcription Factors and Chemical Compounds Targeting the HIF-α Hydroxylases

The hypoxia-inducible transcription factors (HIFs) are central components in the cellular responses to a lack of O(2), i.e. hypoxia. Homologs of the HIF system (HIF-1, -2 and -3) are detectable in all nucleated cells of multicellular organisms. Active HIFs are heterodimers (HIF-alpha/ beta). In hypoxia the O(2)-labile alpha-subunit is translocated to the nucleus where it binds HIF-beta. Over 100 HIF target genes have already been identified. The translational products of these genes increase O(2) delivery to hypoxic tissues, such as erythropoietin which stimulates the production of red blood cells, and they adapt cellular metabolism to hypoxia, such as glycolytic enzymes. HIFs are inactive in normoxia because of O(2)-dependent enzymatic hydroxylation and subsequent degradation of their alpha-subunit. Three HIF-alpha prolyl hydroxylases (PHD1, 2 and 3) initiate proteasomal degradation while an asparaginyl hydroxylase (factor inhibiting HIF-1, FIH-1) inhibits the function of the C-terminal transactivation domain of HIF-alpha. In addition to O(2) and 2-oxoglutarate, the HIF-alpha hydroxylases require Fe(2+) and ascorbate as co-factors. Products of glycolysis can act as endogenous inhibitors of HIF hydroxylases which may lead to sustained activation of HIFs in cancer cells. The cofactor requirements define the routes to inhibition of the enzymes when HIF activation is desirable. In particular, 2-oxoglutarate analogues have emerged as promising tools for stimulation of erythropoiesis and angiogenesis ("HIF-stabilizers"). However, as the HIF system promotes the transcription of many genes, and other 2-oxoglutarate dependent dioxygenases are likely to be inhibited by the same analogues, careful evaluation of the inhibitors seems mandatory prior to their clinical use.

Hypoxia-inducible factors and tubular cell survival in isolated perfused kidneys

Adaptation to hypoxic environment is conferred through hypoxia-inducible transcription factors (HIFs). We have previously shown that the HIF system is transiently activated in vivo in radiocontrast-induced acute renal failure, associated with profound hypoxia in the renal medulla. Medullary thick ascending limbs (mTALs), the most affected nephron segments in this model, were virtually unable to mount an adaptive HIF response. Here, we study correlations between oxygenation, HIF activation, and cell viability in a related ex vivo model, the isolated perfused rat kidney (IPK). In IPKs perfused with cell-free oxygenated medium, severe medullary hypoxic damage developed, affecting 42+/-9% of mTALs in the mid-inner stripe. HIF-1alpha tubular immunostaining was noted with a zonal and tubular pattern largely similar to our findings in vivo: in 34+/-3% of collecting ducts (CDs) within the mid-inner stripe and extensively in the papillary tip, whereas mTALs were all HIF-negative. In IPKs supplemented with RBCs (improved oxygen supply), mTAL damage was totally prevented and CDs' HIF expression was attenuated (22+/-4%). By contrast, although measures designed to reduce medullary hypoxia by decreasing tubular reabsorptive activity (furosemide, ouabain, or high-albumin-non-filtering system) reduced mTAL damage, all paradoxically resulted in increased HIF expression in CDs (51+/-4%), and 17+/-3% of mTALs became immunostained as well. Our data confirm that CDs and mTALs have markedly different HIF responses, which correlate with their viability under hypoxic stress. mTALs transcriptional adaptation occurs within a narrow hypoxic range, and it appears that workload reduction can shift mTALs into this window of opportunity for HIF activation and survival.

Cellular oxygen sensing: Crystal structure of hypoxia-inducible factor prolyl hydroxylase (PHD2).

Cellular and physiological responses to changes in dioxygen levels in metazoans are mediated via the posttranslational oxidation of hypoxia-inducible transcription factor (HIF). Hydroxylation of conserved prolyl residues in the HIF-alpha subunit, catalyzed by HIF prolyl-hydroxylases (PHDs), signals for its proteasomal degradation. The requirement of the PHDs for dioxygen links changes in dioxygen levels with the transcriptional regulation of the gene array that enables the cellular response to chronic hypoxia; the PHDs thus act as an oxygen-sensing component of the HIF system, and their inhibition mimics the hypoxic response. We describe crystal structures of the catalytic domain of human PHD2, an important prolyl-4-hydroxylase in the human hypoxic response in normal cells, in complex with Fe(II) and an inhibitor to 1.7 A resolution. PHD2 crystallizes as a homotrimer and contains a double-stranded beta-helix core fold common to the Fe(II) and 2-oxoglutarate-dependant dioxygenase family, the residues of which are well conserved in the three human PHD enzymes (PHD 1-3). The structure provides insights into the hypoxic response, helps to rationalize a clinically observed mutation leading to familial erythrocytosis, and will aid in the design of PHD selective inhibitors for the treatment of anemia and ischemic disease.

Preconditional Activation of Hypoxia-Inducible Factors Ameliorates Ischemic Acute Renal Failure

Activation of hypoxia-inducible transcription factor (HIF) has been identified as an important mechanism of cellular adaptation to low oxygen. Normoxic degradation of HIF is mediated by oxygen-dependent hydroxylation of specific prolyl residues of the regulative alpha-subunits by HIF prolyl hydroxylases (PHD). It was hypothesized that inhibition of HIF degradation by either hypoxia or pharmacologic inhibition of PHD would confer protection against subsequent ischemic injury. For testing this hypothesis ischemic acute renal failure was induced in rats by 40 min of clamping of the left renal artery after right-sided nephrectomy. Before surgery, pretreatment with either carbon monoxide, leading to tissue hypoxia, or the novel PHD inhibitor FG-4487 was applied. No toxic effects of FG-4487 were observed. Both pretreatments strongly induced the accumulation of HIF-1alpha and HIF-2alpha in tubular and peritubular cells, respectively, as well as HIF target gene expression. The course of subsequent ischemic injury was significantly ameliorated by both strategies of preconditioning, as evident from a significant improvement of serum creatinine and serum urea after 24 and 72 h. Furthermore, tissue injury and apoptosis were less severe, which were quantified by application of a standardized histologic scoring system in a blinded manner. In conclusion, the data provide proof of principle that preconditional activation of the HIF system protects against ischemic injury. Inhibiting the activity of HIF hydroxylases therefore seems to have considerable clinical perspectives.

Redox regulation of the hypoxia-inducible factor

Reactive oxygen species (ROS) have long been considered only as cyto- and genotoxic. However, there is now compelling evidence that ROS also act as second messengers in response to various stimuli, such as growth factors, hormones and cytokines. The hypoxia-inducible transcription factor (HIF) is a master regulator of oxygen-sensitive gene expression. More recently, HIF has also been shown to respond to non-hypoxic stimuli. Interestingly, recent reports indicate that ROS regulate HIF stability and transcriptional activity in well-oxygenated cells, as well as under hypoxic conditions. Consequently, ROS appear to be key players in regulating HIF-dependent pathways under both normal and pathological circumstances. This review summarizes the current understanding of the role of ROS in the regulation of the mammalian HIF system.

Regulation of erythropoietin production

The glycoprotein hormone erythropoietin (EPO) is an essential growth and survival factor for erythroid progenitor cells, and the rate of red blood cell production is normally determined by the serum EPO concentration. EPO production is inversely related to oxygen availability, so that an effective feedback loop is established, which controls erythropoiesis. Since recombinant EPO became available as an effective therapeutic agent, significant progress has also been made in understanding the basis of this feedback control. The main determinant of EPO synthesis is the transcriptional activity of its gene in liver and kidneys, which is related to local oxygen tensions. This control is achieved by hypoxia-inducible transcription factors (HIF), consisting of a constitutive beta-subunit and one of two alternative oxygen-regulated HIFalpha subunits (HIF-1alpha and HIF-2alpha). In the presence of oxygen (normoxia) the HIFalpha subunits are hydroxylated, which targets them for proteasomal degradation. Under hypoxia, because of the lack of molecular oxygen, HIF cannot be hydroxylated and is thereby stabilized. Although HIF-1alpha was the first transcription factor identified through its ability to bind to an enhancer sequence of the EPO gene, more recent evidence suggests that HIF-2alpha is responsible for the regulation of EPO. Although EPO is a prime example for an oxygen- regulated gene, the role of the HIF system goes far beyond the regulation of EPO, because it operates widely in almost all cells and controls a broad transcriptional response to hypoxia, including genes involved in cell metabolism, angiogenesis and vascular tone. Further evidence suggests that apart from its effect as an erythropoietic hormone EPO acts as a paracrine, tissue-protective protein in the brain and possibly also in other organs.

A role for hypoxia and hypoxia-inducible transcription factors in tumor physiology.

Cumulative acquisition of genetic alterations affecting oncogenes or tumor suppressor genes may select for tumor cell clones with enhanced proliferation and survival potential. As a result oxygen and nutrient consumption increases, leading to a tumor microenvironment characterized by low oxygen tension, low glucose levels, and an acidic pH. Hypoxia-inducible transcription factors (HIF) are activated in response to hypoxia, apparently via reduced activity of the recently identified class of 2-oxoglutarate dependent oxygenases, as well as various tumor specific genetic alterations. A widespread HIF activation can be observed in a variety of malignant tumors. The HIF system induces adaptive responses including angiogenesis, glycolysis, and pH regulation which confer increased resistance towards the hostile tumor microenvironment. Apart from protumorigenic the wide-ranging HIF pathway may also have antitumorigenic components, which might, however, be counteracted by specific genetic mechanisms. Thus mounting evidence suggests that the HIF system plays a decisive role in tumor physiology and progression. Moreover, recent insight into this pathway has opened novel and potentially selective therapeutic approaches.