Deficiency of factor-inhibiting HIF creates a tumor-promoting immune microenvironment.

Hypoxia signaling influences tumor development through both cell-intrinsic and -extrinsic pathways. Inhibiting hypoxia-inducible factor (HIF) function has recently been approved as a cancer treatment strategy. Hence, it is important to understand how regulators of HIF may affect tumor growth under physiological conditions. Here we report that in aging mice factor-inhibiting HIF (FIH), one of the most studied negative regulators of HIF, is a haploinsufficient suppressor of spontaneous B cell lymphomas, particular pulmonary B cell lymphomas. FIH deficiency alters immune composition in aged mice and creates a tumor-supportive immune environment demonstrated in syngeneic mouse tumor models. Mechanistically, FIH-defective myeloid cells acquire tumor-supportive properties in response to signals secreted by cancer cells or produced in the tumor microenvironment with enhanced arginase expression and cytokine-directed migration. Together, these data demonstrate that under physiological conditions, FIH plays a key role in maintaining immune homeostasis and can suppress tumorigenesis through a cell-extrinsic pathway.

Molecular characterization and function of hif1a and fih1 in response to acute thermal stress in American shad (Alosa sapidissima).

In order to evaluate the function of hypoxia-inducible factor-1 alpha (hif1α) and factor inhibiting hif1α (fih1) in response to thermal stress, we first conducted a functional analysis of A. sapidissima hif1α and fih1, and determined hif1α and fih1 expressions in different tissues in response to thermal stress based on identified housekeeping genes (HKGs). The results showed that hif1α and fih1 were mainly located in the nucleus and cytoplasm. The full length cDNA sequence of hif1α and fih1 was 4073 bp and 2759 bp, respectively. The cDNA sequence of hif1α includes 15 exons encoding 750 amino acid residues, and the full length cDNA sequence of fih1 contains 9 exons encoding 354 amino acid residues. During the acute thermal stress transferring from 16 ± 0.5 °C (control) to 20 ± 0.5 °C, 25 ± 0.5 °C, and 30 ± 0.5 °C for 15 min, it was found that the expression trends of hif1α and fih1 showed an inhibitory regulation in the heart, while they consistently expressed in brain, intestine, muscle, gill, kidney and liver. In conclusion, this is the first study to identify the tissue-specific HKGs in A. sapidissima and found that ef1α and ß-actin are the most suitable HKGs. Hif1α and Fih1 are mainly the nuclear and cytoplasmic proteins, respectively, having high levels in the heart and brain. Alosa sapidissima countered a temperature increase from 16 to 25 ℃ by regulating the expressions of hif1α and fih1, but their physiological regulatory functions were unable to cope with acute thermal stress when the temperature difference was 14 ℃ (from 16 to 30 ℃).

Factor inhibiting HIF can catalyze two asparaginyl hydroxylations in VNVN motifs of ankyrin fold proteins.

The aspariginyl hydroxylase human factor inhibiting hypoxia-inducible factor (FIH) is an important regulator of the transcriptional activity of hypoxia-inducible factor. FIH also catalyzes the hydroxylation of asparaginyl and other residues in ankyrin repeat domain-containing proteins, including apoptosis stimulating of p53 protein (ASPP) family members. ASPP2 is reported to undergo a single FIH-catalyzed hydroxylation at Asn-986. We report biochemical and crystallographic evidence showing that FIH catalyzes the unprecedented post-translational hydroxylation of both asparaginyl residues in "VNVN" and related motifs of ankyrin repeat domains in ASPPs (i.e., ASPP1, ASPP2, and iASPP) and the related ASB11 and p18-INK4C proteins. Our biochemical results extend the substrate scope of FIH catalysis and may have implications for its biological roles, including in the hypoxic response and ASPP family function.

Biochemical and Structural Insights into FIH-Catalysed Hydroxylation of Transient Receptor Potential Ankyrin Repeat Domains.

Transient receptor potential (TRP) channels have important roles in environmental sensing in animals. Human TRP subfamily A member 1 (TRPA1) is responsible for sensing allyl isothiocyanate (AITC) and other electrophilic sensory irritants. TRP subfamily vanilloid member 3 (TRPV3) is involved in skin maintenance. TRPV3 is a reported substrate of the 2-oxoglutarate oxygenase factor inhibiting hypoxia-inducible factor (FIH). We report biochemical and structural studies concerning asparaginyl hydroxylation of the ankyrin repeat domains (ARDs) of TRPA1 and TRPV3 catalysed by FIH. The results with ARD peptides support a previous report on FIH-catalysed TRPV3 hydroxylation and show that, of the 12 potential TRPA1 sequences investigated, one sequence (TRPA1 residues 322-348) undergoes hydroxylation at Asn336. Structural studies reveal that the TRPA1 and TRPV3 ARDs bind to FIH with a similar overall geometry to most other reported FIH substrates. However, the binding mode of TRPV3 to FIH is distinct from that of other substrates.

High-altitude deer mouse hypoxia-inducible factor-2α shows defective interaction with CREB-binding protein.

Numerous mammalian species have adapted to the chronic hypoxia of high altitude. Recent genomic studies have identified evidence for natural selection of genes and associated genetic changes in these species. A major gap in our knowledge is an understanding of the functional significance, if any, of these changes. Deer mice (Peromyscus maniculatus) live at both low and high altitudes in North America, providing an opportunity to identify functionally important genetic changes. High-altitude deer mice show evidence of natural selection on the Epas1 gene, which encodes for hypoxia-inducible factor-2α (Hif-2α), a central transcription factor of the hypoxia-inducible factor pathway. An SNP encoding for a T755M change in the Hif-2α protein is highly enriched in high-altitude deer mice, but its functional significance is unknown. Here, using coimmunoprecipitation and transcriptional activity assays, we show that the T755M mutation produces a defect in the interaction of Hif-2α with the transcriptional coactivator CREB-binding protein. This results in a loss of function because of decreased transcriptional activity. Intriguingly, the effect of this mutation depends on the amino acid context. Interchanges between methionine and threonine at the corresponding position in house mouse (Mus musculus) Hif-2α are without effects on CREB-binding protein binding. Furthermore, transfer of a set of deer mouse-specific Hif-2α amino acids to house mouse Hif-2α is sufficient to confer sensitivity of house mouse Hif-2α to the T755M substitution. These findings provide insight into high-altitude adaptation in deer mice and evolution at the Epas1 locus.

Asparagine Hydroxylation is a Reversible Post-translational Modification.

Amino acid hydroxylation is a common post-translational modification, which generally regulates protein interactions or adds a functional group that can be further modified. Such hydroxylation is currently considered irreversible, necessitating the degradation and re-synthesis of the entire protein to reset the modification. Here we present evidence that the cellular machinery can reverse FIH-mediated asparagine hydroxylation on intact proteins. These data suggest that asparagine hydroxylation is a flexible and dynamic post-translational modification akin to modifications involved in regulating signaling networks, such as phosphorylation, methylation and ubiquitylation.

Nuclear entry and export of FIH are mediated by HIF1α and exportin1, respectively.

Hypoxia plays a crucial role at cellular and physiological levels in all animals. The responses to chronic hypoxia are, at least substantially, orchestrated by activation of the hypoxia inducible transcription factors (HIFs), whose stability and subsequent transcriptional activation are regulated by HIF hydroxylases. Factor inhibiting HIF (FIH), initially isolated as a HIFα interacting protein following a yeast two-hybrid screen, is an asparaginyl hydroxylase that negatively regulates transcriptional activation by HIF. This study aimed to define the mechanisms that govern transitions of FIH between the nucleus and cytoplasm. We report that FIH accumulates in the nucleus within a short time window during hypoxia treatment. We provide evidence, based on the application of genetic interventions and small molecule inhibition of the HIF hydroxylases, that the nuclear localization of FIH is governed by two opposing processes: nuclear entry by 'coupling' with HIF1α for importin ß1-mediated nuclear import and active export via a Leptomycin B-sensitive exportin1-dependent pathway.This article has an associated First Person interview with the first author of the paper.

Role of Microtubule-Associated Factors in HIF1α Nuclear Translocation.

Adaptation to hypoxia is essential for regulating the survival and functions of hypoxic cells; it is mainly mediated by the hypoxia-inducible factor 1 (HIF1). The alpha subunit of HIF1 (HIF1α) is a well-known regulatory component of HIF1, which is tightly controlled by various types of HIF1α-regulating processes. Previous research has shown that microtubule-regulated HIF1α nuclear translocation is a key factor for HIF1 activation under hypoxia. In this review, we summarize experimental reports on the role of microtubule-associated factors, such as microtubule, dynein, and dynein adaptor protein, in nuclear translocation of HIF1α. Based upon scientific evidence, we propose a bicaudal D homolog (BICD) as a novel HIF1α translocation regulating factor. A deeper understanding of the mechanism of the action of regulatory factors in controlling HIF1α nuclear translocation will provide novel insights into cell biology under hypoxia.

Deletion of the fih gene encoding an inhibitor of hypoxia-inducible factors increases hypoxia tolerance in zebrafish.

Many aerobic organisms have developed molecular mechanism to tolerate hypoxia, but the specifics of these mechanisms remain poorly understood. It is important to develop genetic methods that confer increased hypoxia tolerance to intensively farmed aquatic species, as these are maintained in environments with limited available oxygen. As an asparaginyl hydroxylase of hypoxia-inducible factors (HIFs), factor inhibiting HIF (FIH) inhibits transcriptional activation of hypoxia-inducible genes by blocking the association of HIFs with the transcriptional coactivators CREB-binding protein (CBP) and p300. Therefore, here we sought to test whether fih is involved in regulating hypoxia tolerance in the commonly used zebrafish model. Overexpressing the zebrafish fih gene in epithelioma papulosum cyprini (EPC) cells and embryos, we found that fih inhibits the transcriptional activation of zebrafish HIF-α proteins. Using CRISPR/Cas9 to obtain fih-null zebrafish mutants, we noted that the fih deletion makes zebrafish more tolerant of hypoxic conditions than their WT siblings, but does not result in oxygen consumption rates that significantly differ from those of WT fish. Of note, we identified fewer apoptotic cells in adult fih-null zebrafish brains and in fih-null embryos, possibly explaining why the fih-null mutant had greater hypoxia tolerance than the WT. Moreover, the fih deletion up-regulated several hypoxia-inducible genes in fih-null zebrafish exposed to hypoxia. The findings of our study suggest that fih plays a role in hypoxia tolerance by affecting the rate of cellular apoptosis in zebrafish.

Molecular characterization and expression regulation of the factor-inhibiting HIF-1 (FIH-1) gene under hypoxic stress in bighead carp (Aristichthys nobilis).

Factor-inhibiting HIF-1 (FIH-1) is an asparagine hydroxylase that interacts with hypoxia-inducible factor 1α (HIF-1α) to regulate transcriptional activity of HIF-1. Few studies of fish FIH-1 have been reported to date. In this study, the cDNA of FIH-1 gene was cloned and characterized for bighead carp, Aristichthys nobilis (AnFIH-1). The AnFIH-1 cDNA is 2065 bp in length, encoding a protein of 357 amino acid (aa) residues, which contains a JmjC homology region of the jumonji transcription factors. AnFIH-1 shares high identities with other vertebrate FIH-1 (79.1-96.4%), especially in the JmjC homology region, suggesting its conserved function. During the embryonic stages of A. nobilis, AnFIH-1 had significantly high expression levels in unfertilized egg and blastula. In healthy tissues, its predominant mRNA expression was detected in muscle. The mRNA levels of AnFIH-1 were significantly upregulated in the liver, gill, hypothalamus, and spleen after hypoxic treatment, and then decreased to pretreatment levels after 6-h re-oxygenation. However, in the muscle, continual increasing of mRNA expression was observed after hypoxic shock and re-oxygenation. These results indicate that FIH-1 may play an important role in physiological regulation for adapting to hypoxia stress in A. nobilis.

Ferritin heavy chain controls the HIF-driven hypoxic response by activating the asparaginyl hydroxylase FIH.

Hypoxia-inducible factor 1 (HIF-1) is a key player in cellular response to hypoxia. The stability and transcriptional activity of this protein are oxygen-dependently regulated by the prolyl hydroxylases PHD1-3 and the asparaginyl hydroxylase FIH. Recently, ferritin heavy chain (FTH1) has been characterized to reinforce the HIF-1 signaling pathway in an indirect way through the inhibition of PHD activity by depleting the free iron pool in the cytoplasm. In the present study, we addressed the role of FTH1 in the FIH control of HIF-1 activity. Unexpectedly, immunoprecipitation analyses revealed that FTH1 directly interacted with FIH. In an in vitro hydroxylation assay, FTH1 was found to facilitate the FIH-mediated Asn803 hydroxylation in HIF-1α. As expected, FTH1 prevented the recruitment of p300 to HIF-1α through the Asn803 hydroxylation. In luciferase reporter analyses, FTH1 was found to repress the transcriptional activity of HIF-1α in HCT116 cells under either normoxic or hypoxic conditions. Consequently, FTH1 downregulated the expression of the HIF-1 target genes, such as VEGF, CA9 and GLUT1. Our results suggest a new role of FTH1 as a co-regulator for the FIH-mediated oxygen sensing pathway. Since HIF-1α is involved in pathogenesis of diverse hypoxia-associated diseases, we propose that FTH1 be a potential target in developing new therapeutic strategies against such diseases.

Ankyrin Repeat Proteins of Orf Virus Influence the Cellular Hypoxia Response Pathway.

Hypoxia-inducible factor (HIF) is a transcriptional activator with a central role in regulating cellular responses to hypoxia. It is also emerging as a major target for viral manipulation of the cellular environment. Under normoxic conditions, HIF is tightly suppressed by the activity of oxygen-dependent prolyl and asparaginyl hydroxylases. The asparaginyl hydroxylase active against HIF, factor inhibiting HIF (FIH), has also been shown to hydroxylate some ankyrin repeat (ANK) proteins. Using bioinformatic analysis, we identified the five ANK proteins of the parapoxvirus orf virus (ORFV) as potential substrates of FIH. Consistent with this prediction, coimmunoprecipitation of FIH was detected with each of the ORFV ANK proteins, and for one representative ORFV ANK protein, the interaction was shown to be dependent on the ANK domain. Immunofluorescence studies revealed colocalization of FIH and the viral ANK proteins. In addition, mass spectrometry confirmed that three of the five ORFV ANK proteins are efficiently hydroxylated by FIH in vitro While FIH levels were unaffected by ORFV infection, transient expression of each of the ORFV ANK proteins resulted in derepression of HIF-1α activity in reporter gene assays. Furthermore, ORFV-infected cells showed upregulated HIF target gene expression. Our data suggest that sequestration of FIH by ORFV ANK proteins leads to derepression of HIF activity. These findings reveal a previously unknown mechanism of viral activation of HIF that may extend to other members of the poxvirus family. IMPORTANCE: The protein-protein binding motif formed from multiple repeats of the ankyrin motif is common among chordopoxviruses. However, information on the roles of these poxviral ankyrin repeat (ANK) proteins remains limited. Our data indicate that the parapoxvirus orf virus (ORFV) is able to upregulate hypoxia-inducible factor (HIF) target gene expression. This response is mediated by the viral ANK proteins, which sequester the HIF regulator FIH (factor inhibiting HIF). This is the first demonstration of any viral protein interacting directly with FIH. Our data reveal a new mechanism by which viruses reprogram HIF, a master regulator of cellular metabolism, and also show a new role for the ANK family of poxvirus proteins.

Radioprotective Activity and Preliminary Mechanisms of N-oxalyl-d-phenylalanine (NOFD) In Vitro.

The radiation-induced damage to the human body is primarily caused by excessive reactive oxygen species (ROS) production after irradiation. Therefore, the removal of the increase of ROS caused by ionizing radiation (IR) has been the focus of research on radiation damage protective agents. Hypoxia inducible factor (HIF) is a transcription factor in human and plays an important role in regulating the body metabolism. Factor inhibiting HIF (FIH) is an endogenous inhibitor factor of HIF protein under normoxia conditions. It has been shown that the high expression of HIF protein has a certain repair effect on radiation-induced intestinal injury and hematopoietic system damage in mice; however, it is not clear about the effect of HIF on the level of ROS after radiation. In this study, the role of N-oxalyl-d-phenylalanine (NOFD), an FIH inhibitor, for its effect on alleviating ROS level is investigated in the cells. Our results indicate that pretreatment with NOFD can mitigate ROS level and alleviate IR-induced DNA damage and apoptosis in vitro. Therefore, HIF can be used as a target on scavengers. Furthermore, in order to explore the relevant mechanism, we also test the expression of relevant HIF downstream genes in the cells, finding that Notch-2 gene is more sensitive to NOFD treatment. This experiment result is used to support the subsequent mechanism experiments.

Kinetic Investigations of the Role of Factor Inhibiting Hypoxia-inducible Factor (FIH) as an Oxygen Sensor.

The hypoxia-inducible factor (HIF) hydroxylases regulate hypoxia sensing in animals. In humans, they comprise three prolyl hydroxylases (PHD1-3 or EGLN1-3) and factor inhibiting HIF (FIH). FIH is an asparaginyl hydroxylase catalyzing post-translational modification of HIF-α, resulting in reduction of HIF-mediated transcription. Like the PHDs, FIH is proposed to have a hypoxia-sensing role in cells, enabling responses to changes in cellular O2 availability. PHD2, the most important human PHD isoform, is proposed to be biochemically/kinetically suited as a hypoxia sensor due to its relatively high sensitivity to changes in O2 concentration and slow reaction with O2. To ascertain whether these parameters are conserved among the HIF hydroxylases, we compared the reactions of FIH and PHD2 with O2. Consistent with previous reports, we found lower Km(app)(O2) values for FIH than for PHD2 with all HIF-derived substrates. Under pre-steady-state conditions, the O2-initiated FIH reaction is significantly faster than that of PHD2. We then investigated the kinetics with respect to O2 of the FIH reaction with ankyrin repeat domain (ARD) substrates. FIH has lower Km(app)(O2) values for the tested ARDs than HIF-α substrates, and pre-steady-state O2-initiated reactions were faster with ARDs than with HIF-α substrates. The results correlate with cellular studies showing that FIH is active at lower O2 concentrations than the PHDs and suggest that competition between HIF-α and ARDs for FIH is likely to be biologically relevant, particularly in hypoxic conditions. The overall results are consistent with the proposal that the kinetic properties of individual oxygenases reflect their biological capacity to act as hypoxia sensors.

The factor inhibiting HIF regulates T cell differentiation and anti-tumour efficacy.

T cells must adapt to variations in tissue microenvironments; these adaptations include the degree of oxygen availability. The hypoxia-inducible factor (HIF) transcription factors control much of this adaptation, and thus regulate many aspects of T cell activation and function. The HIFs are in turn regulated by oxygen-dependent hydroxylases: both the prolyl hydroxylases (PHDs) which interact with the VHL tumour suppressor and control HIF turnover, and the asparaginyl hydroxylase known as the Factor inhibiting HIF (FIH), which modulates HIF transcriptional activity. To determine the role of this latter factor in T cell function, we generated T cell-specific FIH knockout mice. We found that FIH regulates T cell fate and function in a HIF-dependent manner and show that the effects of FIH activity occur predominantly at physiological oxygen concentrations. T cell-specific loss of FIH boosts T cell cytotoxicity, augments T cell expansion in vivo, and improves anti-tumour immunotherapy in mice. Specifically inhibiting FIH in T cells may therefore represent a promising strategy for cancer immunotherapy.

Oxomer- and Reporter Gene-Based Analysis of FIH Activity in Cells.

Cellular and tissue adaptations to oxygen deprivation (hypoxia) are necessary for both normal physiology and disease. Responses to hypoxia are initiated by the cellular oxygen sensors prolyl-4-hydroxylase domain (PHD) proteins 1-3 and factor inhibiting HIF (FIH). These enzymes regulate the transcription factor hypoxia-inducible factor (HIF) in a hypoxia-sensitive manner. FIH also regulates proteins outside the HIF pathway, including the deubiquitinase OTUB1. Numerous preclinical analyses have demonstrated that treatment with HIF hydroxylase inhibitors is beneficial and protective in many hypoxia-associated diseases. However, clinically available HIF hydroxylase inhibitors increase erythropoietin (EPO) gene expression and red blood cell production, which can be detrimental in hypoxia-associated conditions, such as ischemia/reperfusion injury of the heart or chronic inflammation. Our understanding of the relevance of FIH in (patho)physiology is only in its infancy, but FIH activity does not govern erythropoietin expression. Therefore, it is of prime interest to assess the relevance of FIH activity in (patho)physiology in detail, as it may contribute to developing novel therapeutic options for treating hypoxia-associated diseases that do not affect Epo regulation. Here, we describe specific protocols for two different methods to assess FIH enzymatic activity within cells, using a HIF-dependent firefly luciferase-reporter gene and an oxomer-dependent assay. Oxomers are oxygen-dependent stable protein oligomers formed by FIH, for example, with the deubiquitinase OTUB1. Oxomer formation directly depends on FIH activity, providing a suitable cellular readout for an easy-to-use analysis of FIH enzymatic activity in cellulo. These techniques permit an analysis of FIH activity toward HIF and outside the HIF pathway, allowing the investigation of FIH activity under different (patho)physiological conditions and assessment of novel (putative) inhibitors.

Biochemistry of the hypoxia-inducible factor hydroxylases.

The hypoxia-inducible factors are α,ß-heterodimeric transcription factors that mediate the chronic response to hypoxia in humans and other animals. Protein hydroxylases belonging to two different structural subfamilies of the Fe(II) and 2-oxoglutarate (2OG)-dependent oxygenase superfamily modify HIFα. HIFα prolyl-hydroxylation, as catalysed by the PHDs, regulates HIFα levels and, consequently, α,ß-HIF levels. HIFα asparaginyl-hydroxylation, as catalysed by factor inhibiting HIF (FIH), regulates the transcriptional activity of α,ß-HIF. The activities of the PHDs and FIH are regulated by O2 availability, enabling them to act as hypoxia sensors. We provide an overview of the biochemistry of the HIF hydroxylases, discussing evidence that their kinetic and structural properties may be tuned to their roles in the HIF system. Avenues for future research and therapeutic modulation are discussed.

Measurement of kinetic isotope effects on peptide hydroxylation using MALDI-MS.

Primary kinetic isotope effects (KIEs) provide unique insight into enzymatic reactions, as they can reveal rate-limiting steps and detailed chemical mechanisms. HIF hydroxylases, part of a family of 2-oxoglutarate (2OG) oxygenases are central to the regulation of many crucial biological processes through O2-sensing, but present a challenge to monitor due to the large size of the protein substrate and the similarity between native and hydroxylated substrate. MALDI-TOF MS is a convenient tool to measure peptide masses, which can also be used to measure the discontinuous kinetics of peptide hydroxylation for Factor Inhibiting HIF (FIH). Using this technique, rate data can be observed from the mole-fraction of CTAD and CTAD-OH in small volumes, allowing noncompetitive H/D KIEs to be measured. Slow dCTAD substrate leads to extensive uncoupling of O2 consumption from peptide hydroxylation, leading to enzyme autohydroxylation, which is observed using UV-vis spectroscopy. Simultaneously measuring both the normal product, CTAD-OH, and the uncoupled product, autohydroxylated enzyme, the KIE on the microscopic step of hydrogen atom transfer (HAT) can be estimated. MALDI-MS analysis is a strong method for monitoring reactions that hydroxylate peptides, and can be generalized to other similar reactions, and simultaneous kinetic detection of branched products can provide valuable insight on microscopic KIEs at intermediate mechanistic steps.

Differential methylation in EGLN1 associates with blood oxygen saturation and plasma protein levels in high-altitude pulmonary edema.

BACKGROUND: High-altitude (HA, 2500 m) hypoxic exposure evokes a multitude of physiological processes. The hypoxia-sensing genes though influence transcriptional output in disease susceptibility; the exact regulatory mechanisms remain undetermined in high-altitude pulmonary edema (HAPE). Here, we investigated the differential DNA methylation distribution in the two genes encoding the oxygen-sensing HIF-prolyl hydroxylases, prolyl hydroxylase domain protein 2 (PHD2) and factor inhibiting HIF-1α and the consequent contributions to the HAPE pathophysiology. METHODS: Deep sequencing of the sodium bisulfite converted DNA segments of the two genes, Egl nine homolog 1 (EGLN1) and Hypoxia Inducible Factor 1 Subunit Alpha Inhibitor (HIF1AN), was conducted to analyze the differential methylation distribution in three study groups, namely HAPE-patients (HAPE-p), HAPE-free sojourners (HAPE-f) and healthy HA natives (HLs). HAPE-p and HAPE-f were permanent residents of low altitude (< 200 m) of North India who traveled to Leh (3500 m), India, and were recruited through Sonam Norboo Memorial (SNM) hospital, Leh. HLs were permanent residents of altitudes at and above 3500 m. In addition to the high resolution, bisulfite converted DNA sequencing, gene expression of EGLN1 and HIF1AN and their plasma protein levels were estimated. RESULTS: A significantly lower methylation distribution of CpG sites was observed in EGLN1 and higher in HIF1AN (P < 0.01) in HAPE-p compared to the two control groups, HAPE-f and HLs. Of note, differential methylation distribution of a few CpG sites, 231,556,748, 231,556,804, 231,556,881, 231,557,317 and 231,557,329, in EGLN1 were significantly associated with the risk of HAPE (OR = 4.79-10.29; P = 0.048-004). Overall, the methylation percentage in EGLN1 correlated with upregulated plasma PHD2 levels (R = - 0.36, P = 0.002) and decreased peripheral blood oxygen saturation (SpO2) levels (R = 0.34, P = 0.004). We also identified a few regulatory SNPs in the DNA methylation region of EGLN1 covering chr1:231,556,683-231,558,443 suggestive of the functional role of differential methylation distribution of these CpG sites in the regulation of the genes and consequently in the HIF-1α signaling. CONCLUSIONS: Significantly lower methylation distribution in EGLN1 and the consequent physiological influences annotated its functional epigenetic relevance in the HAPE pathophysiology.

Feedback of hypoxia-inducible factor-1alpha (HIF-1alpha) transcriptional activity via redox factor-1 (Ref-1) induction by reactive oxygen species (ROS).

Hypoxia-inducible factor-1alpha (HIF-1alpha) is important for adaptation to hypoxia. Hypoxia is a common feature of cancer and inflammation, by which HIF-1alpha increases. However, prolonged hypoxia decreases HIF-1alpha, and the underlying mechanisms currently remain unclear. Cellular reactive oxygen species (ROS) increases in cancer and inflammation. In the present study, we demonstrated that prolonged hypoxia increased ROS, which induced prolyl hydroxylase domain-containing protein 2 (PHD2) and factor inhibiting HIF-1 (FIH-1), major regulators of HIF-1alpha. Cellular stress response (CSR) increased HIF-1alpha transcriptional activity by scavenging endogenous ROS. PHD2 and FIH-1 were induced by external hydrogen peroxide (H2O2) but were suppressed by ROS-scavenging catalase. We investigated the mechanisms by which PHD2 and FIH-1 are regulated by ROS. The knockdown of HIF-1alpha decreased PHD2 and FIH-1 mRNA levels, suggesting their regulation by HIF-1alpha. We then focused on redox factor-1 (Ref-1), which is a regulator of HIF-1alpha transcriptional activity. The knockdown of Ref-1 decreased PHD2 and FIH-1. Ref-1 was regulated by ROS. Prolonged hypoxia and the addition of H2O2 induced the expression of Ref-1. Furthermore, the knockdown of p65, a component of kappa-light-chain enhancer of activated B cells (NF-κB), efficiently inhibited the induction of Ref-1 by ROS. Collectively, the present results showed that prolonged hypoxia or increased ROS levels induced Ref-1, leading to the activation of HIF-1alpha transcriptional activity, while the activation of HIF-1alpha via Ref-1 induced PHD2 and FIH-1, causing the feedback of HIF-1alpha. To the best of our knowledge, this is the first study to demonstrate the regulation of HIF-1alpha via Ref-1 by ROS.