Factor Inhibiting HIF Research Articles

Evolutionary Conserved Regulation of HIF-1β by NF-κB

Hypoxia Inducible Factor-1 (HIF-1) is essential for mammalian development and is the principal transcription factor activated by low oxygen tensions. HIF-α subunit quantities and their associated activity are regulated in a post-translational manner, through the concerted action of a class of enzymes called Prolyl Hydroxylases (PHDs) and Factor Inhibiting HIF (FIH) respectively. However, alternative modes of HIF-α regulation such as translation or transcription are under-investigated, and their importance has not been firmly established. Here, we demonstrate that NF-κB regulates the HIF pathway in a significant and evolutionary conserved manner. We demonstrate that NF-κB directly regulates HIF-1β mRNA and protein. In addition, we found that NF-κB–mediated changes in HIF-1β result in modulation of HIF-2α protein. HIF-1β overexpression can rescue HIF-2α protein levels following NF-κB depletion. Significantly, NF-κB regulates HIF-1β (tango) and HIF-α (sima) levels and activity (Hph/fatiga, ImpL3/ldha) in Drosophila, both in normoxia and hypoxia, indicating an evolutionary conserved mode of regulation. These results reveal a novel mechanism of HIF regulation, with impact in the development of novel therapeutic strategies for HIF–related pathologies including ageing, ischemia, and cancer.

Tumor hypoxia and genetic alterations in sporadic cancers

The cancer genome contains many gene alterations. How cancer cells acquire these alterations is a matter for discussion. One hypothesis is that cancer cells obtain mutations in genome stability genes at an early stage of tumor development, which results in genetic instability and generates a gene pool that enhances cellular proliferation and survival. Another hypothesis puts its emphasis on the natural selection of gene mutations for fitness. Recent data for systematic cancer genome sequencing shows that mutations in stability genes are rare in human sporadic cancers. Instead, many “passenger” mutations that do not drive the carcinogenesis process have been found in the cancer genome. Both the hypotheses mentioned above fall short in explaining recent data. Recently, many studies demonstrate the role of the tumor microenvironment, especially hypoxia and reoxygenation, in genetic instability. In this review, literature will be presented which supports a third hypothesis, i.e. that hypoxia/re-oxygenation induces genetic instability.

At the crossroads of cancer and inflammation: Ras rewires an HIF-driven IL-1 autocrine loop

Malignant tumors of the central nervous system are the leading cause of cancer death for people under 35 years of age. Glioblastoma multiforme (GBM), the most malignant brain tumor in adults is characterized histologically by pseudopalisading necrosis and abnormal vascular development (Fig. 1). These tumors show infiltration by inflammatory cells and the occurrence of hypoxia (lower than physiological O2 levels) in the pseudopalisading cells surrounding the micronecrotic areas. Microenvironmental hypoxia is not restricted to regions adjacent to the large central necrotic areas of gliomas; in fact, it is present in close proximity to the leading/actively growing edge of GBM, where the tumor is actively expanding radially [1]. The hypoxic cells are active participants in the tumor biology and are refractive to conventional therapies, contributing thus to poor patient survival. Currently, the life expectancy for GBM patients is 1–2 years, stressing the need to better understand the signaling mechanisms that underlie their aggressive growth so that new therapies can be designed. Adaptive changes by which cells respond to hypoxia are mediated largely by the transcription factor hypoxia-inducible factor 1 (HIF-1), which transactivates genes whose products play a role in all hallmarks of cancer and is a major target for anticancer therapy [2]. This is evidenced in GBM by the strong expression of HIF-1α on pseudopalisading cells, which activates the mRNA expression for vascular endothelial growth factor (VEGF), a major inducer of tumor angiogenesis (Fig. 1). HIF-1 is a heterodimeric transcription factor composed of the constitutively expressed HIF-1β and the O2-regulated HIF-1α subunits. The protein levels and activation of the α subunit are tightly controlled by two independent but co-regulated post-translational mechanisms [2]. A family of prolylhydroxylases (PHDs) hydroxylates HIF-1α in the presence of O2. This modification allows binding of the tumor suppressor Von Hippel–Lindau, the substrate recognition unit of an E3 ubiquitin ligase complex, polyubiquitylation, and degradation in the proteasome [3]. Factor-inhibiting HIF (FIH), an asparagine hydroxylase, controls transcriptional activity of HIF-1α by modifying a critical asparagine in the C-terminal activation domain. This modification is also O2-dependent and prevents interaction with the transcriptional co-activators p300/CBP. In the absence of O2, both PHDs and FIH are inactive; HIF accumulates in transcriptionally active form and transactivates hypoxia-inducible genes via binding to the hypoxiaresponsive elements (HREs) in their regulatory regions (Fig. 2). Although the availability of themolecular O2 is the primary physiological regulator of HIF-1 through PHDs and FIH, there is a number of other stimuli (cellular oncogenes, loss of tumor suppressor phosphatase and tensin homolog (PTEN), growth factors, cytokines, vascular hormones, viral proteins) that can lead to induction and activation of HIF under normoxic conditions [4]. In this case, enhanced translation of HIF-1α, mediated via activation of cellular signaling pathways (Ras and PI3-Kinase) overcomes the limiting hydroxS. Kaluz : E. G. Van Meir (*) Laboratory of Molecular Neuro-Oncology, Department of Neurosurgery, School of Medicine and Winship Cancer Institute, Emory University, Atlanta, GA, USA e-mail: evanmei@emory.edu

Consequences of IkappaB alpha hydroxylation by the factor inhibiting HIF (FIH)

The factor inhibiting HIF- 1 (FIH-1) hydroxylates many ankyrin repeat-containing proteins including IκBα. It is widely speculated that hydroxylation of IκBα has functional consequences, but the effects of hydroxylation have not been demonstrated. We prepared hydroxylated IκBα and compared it to the unhydroxylated protein. Urea denaturation and amide H/D exchange experiments showed no change in the “foldedness” upon hydroxylation. Surface plasmon resonance measurements of binding to NFκB showed no difference in the NFκB binding kinetics or thermodynamics. Ubiquitin-independent proteasomal degradation experiments showed no difference in the half-life of the protein. Thus, it appears that hydroxylation of IκBα by FIH-1 is inconsequential, at least for the functions we could assay in vitro. Structured summary MINT- 8051494: NF-kappa-B p65 (uniprotkb: Q04207) physically interacts (MI: 0915) with NF-kappa-B p50 (uniprotkb: P25799) and I-kappa-B alpha (uniprotkb: O15111) by surface plasmon resonance (MI: 0107)

Hypoxia-dependent sequestration of an oxygen sensor by a widespread structural motif can shape the hypoxic response - a predictive kinetic model

BackgroundThe activity of the heterodimeric transcription factor hypoxia inducible factor (HIF) is regulated by the post-translational, oxygen-dependent hydroxylation of its α-subunit by members of the prolyl hydroxylase domain (PHD or EGLN)-family and by factor inhibiting HIF (FIH). PHD-dependent hydroxylation targets HIFα for rapid proteasomal degradation; FIH-catalysed asparaginyl-hydroxylation of the C-terminal transactivation domain (CAD) of HIFα suppresses the CAD-dependent subset of the extensive transcriptional responses induced by HIF. FIH can also hydroxylate ankyrin-repeat domain (ARD) proteins, a large group of proteins which are functionally unrelated but share common structural features. Competition by ARD proteins for FIH is hypothesised to affect FIH activity towards HIFα; however the extent of this competition and its effect on the HIF-dependent hypoxic response are unknown.ResultsTo analyse if and in which way the FIH/ARD protein interaction affects HIF-activity, we created a rate equation model. Our model predicts that an oxygen-regulated sequestration of FIH by ARD proteins significantly shapes the input/output characteristics of the HIF system. The FIH/ARD protein interaction is predicted to create an oxygen threshold for HIFα CAD-hydroxylation and to significantly sharpen the signal/response curves, which not only focuses HIFα CAD-hydroxylation into a defined range of oxygen tensions, but also makes the response ultrasensitive to varying oxygen tensions. Our model further suggests that the hydroxylation status of the ARD protein pool can encode the strength and the duration of a hypoxic episode, which may allow cells to memorise these features for a certain time period after reoxygenation.ConclusionsThe FIH/ARD protein interaction has the potential to contribute to oxygen-range finding, can sensitise the response to changes in oxygen levels, and can provide a memory of the strength and the duration of a hypoxic episode. These emergent properties are predicted to significantly shape the characteristics of HIF activity in animal cells. We argue that the FIH/ARD interaction should be taken into account in studies of the effect of pharmacological inhibition of the HIF-hydroxylases and propose that the interaction of a signalling sensor with a large group of proteins might be a general mechanism for the regulation of signalling pathways.

Abnormalities in Oxygen Sensing Define Early and Late Onset Preeclampsia as Distinct Pathologies

BackgroundThe pathogenesis of preeclampsia, a serious pregnancy disorder, is still elusive and its treatment empirical. Hypoxia Inducible Factor-1 (HIF-1) is crucial for placental development and early detection of aberrant regulatory mechanisms of HIF-1 could impact on the diagnosis and management of preeclampsia. HIF-1α stability is controlled by O2-sensing enzymes including prolyl hydroxylases (PHDs), Factor Inhibiting HIF (FIH), and E3 ligases Seven In Absentia Homologues (SIAHs). Here we investigated early- (E-PE) and late-onset (L-PE) human preeclamptic placentae and their ability to sense changes in oxygen tension occurring during normal placental development.Methods and FindingsExpression of PHD2, FIH and SIAHs were significantly down-regulated in E-PE compared to control and L-PE placentae, while HIF-1α levels were increased. PHD3 expression was increased due to decreased FIH levels as demonstrated by siRNA FIH knockdown experiments in trophoblastic JEG-3 cells. E-PE tissues had markedly diminished HIF-1α hydroxylation at proline residues 402 and 564 as assessed with monoclonal antibodies raised against hydroxylated HIF-1α P402 or P564, suggesting regulation by PHD2 and not PHD3. Culturing villous explants under varying oxygen tensions revealed that E-PE, but not L-PE, placentae were unable to regulate HIF-1α levels because PHD2, FIH and SIAHs did not sense a hypoxic environment.ConclusionDisruption of oxygen sensing in E-PE vs. L-PE and control placentae is the first molecular evidence of the existence of two distinct preeclamptic diseases and the unique molecular O2-sensing signature of E-PE placentae may be of diagnostic value when assessing high risk pregnancies and their severity.

Negative Regulation Of HIF-1 Through FIH And SIRT6 In Elite Athletes And With Training

Hypoxia inducible factor 1α (HIF-1α) is a hypoxia sensitive transcription factor suggested to be involved in skeletal muscle adaptation to endurance training. HIF-1α is stabilized in response to an acute bout of exercise, but there is debate regarding the HIF-1 system in response to long-term training. HIF-1 activity is mainly regulated through hydroxylation by prolyl hydroxylases, (PHD1-3), leading to degradation. We have previously shown that PHD2 is the dominant prolyl hydroxylase in human skeletal muscle and that the levels are higher in elite athletes compared to sedentary individuals, while the response gene pyruvate dehydrogenase kinase 1 (PDK-1) is lower. PDK-1 inhibits the activity of pyruvate dehydrogenase, the limiting step for aerobic metabolism. Both factor inhibiting HIF (FIH) and SIRT6 inhibit the transcriptional activity of HIF-1, FIH through inhibition of cofactor binding and SIRT6 as a corepressor through deacetylation of histone 3 lysine 9 of several of the HIF-1 response genes. PURPOSE: To elucidate the role of HIF-1 in endurance-training adapted human skeletal muscle. METHODS: Skeletal muscle biopsies were obtained at rest from the vastus lateralis muscle of 12 males with high oxidative capacity and 9 sedentary controls, as well as from eight sedentary males before and after four weeks of one-legged bicycle training. RESULTS: In elite athletes, the mRNA and protein levels of FIH are seven and four times higher than in control subjects respectively (p<0.05). SIRT6 expression is also seven times higher (p<0.05) in elite athletes. Similar trends are seen in response to four weeks of one-legged endurance training, with 45 % and 35 % increases in expression of FIH (p=0.07) and SIRT6 (p=0.08) respectively. CONCLUSIONS: Elite athletes have significantly higher levels of FIH and SIRT6 than the sedentary control group, and the trend seen in response to only four weeks of training indicate that this is a training-induced response. In combination with previously reported data of higher PHD2 and lower PDK-1 levels in elite athletes, these data suggest an attenuated HIF-1 response with long-term training and an important switch from glycolysis in favor of increased mitochondrial activity.

Expression of nuclear FIH independently predicts overall survival of clear cell renal cell carcinoma patients

Aim The hypoxia inducible factor (HIF) pathway plays an important role in sporadic clear cell renal cell carcinoma (ccRCC) by stimulating processes of angiogenesis, cell proliferation, cell survival and metastases formation. Herein, we evaluate the significance of upstream proteins directly regulating the HIF pathway; the prolyl hydroxylases domain proteins (PHD)1, 2 and 3 and factor-inhibiting HIF (FIH), as prognostic markers for ccRCC. Methods Immunohistochemical marker expression was examined on a tissue microarray containing tumour tissue derived from 100 patients who underwent nephrectomy for ccRCC. Expression levels of HIF, FIH and PHD1, 2 and 3 were correlated with overall survival (OS) and clinicopathological prognostic factors. Results HIF-1α was positively correlated with HIF-2α ( p < 0.0001), PHD1 ( p = 0.024), PHD2 ( p < 0.0001), PHD3 ( p = 0.004), FIH ( p < 0.0001) and VHL ( p = 0.031). HIF-2α levels were significantly associated with FIH ( p < 0.0001) and PHD2 ( p = 0.0155). Mutations in the VHL gene, expression variations of HIF-1α, HIF-2α and PHD1, 2, 3 did not show a correlation to OS or clinicopathological prognostic factors. Tumour stage, grade, diameter, metastastic disease and intensity of nuclear FIH were significantly correlated to OS in univariable analysis ( p = 0.023). Low nuclear FIH levels remained a strong independent prognostic factor in multivariable analysis ( p = 0.009). Conclusion These results show that low nuclear expression of FIH is a strong independent prognostic factor for a poor overall survival in ccRCC.

Structural basis for binding of cyclic 2-oxoglutarate analogues to factor-inhibiting hypoxia-inducible factor

Aromatic analogues of the 2-oxoglutarate co-substrate of the hypoxia-inducible factor hydroxylases are shown to bind at the active site iron: Pyridine-2,4-dicarboxylate binds as anticipated with a single molecule chelating the iron in a bidentate manner. The binding mode of a hydroxamic acid analogue, at least in the crystalline state, is unusual because two molecules of the inhibitor are observed at the active site and partial displacement of the iron binding aspartyl residue was observed.

Force field design and molecular dynamics simulations of factor-inhibiting HIF-1 and its complex with known inhibitors: Implications for rational inhibitor design

Based on molecular dynamics simulations in aqueous solution, we investigate the dynamic properties of factor-inhibiting HIF-1 (FIH1) and its complexes with the substrate 2-oxoglutarate (2OG) and the two known inhibitors, N-oxalylglycine (NOG) and N-oxalyl- d-phenylalanine (NODP). The results obtained with the newly developed force field parameters for the coordination environment of the active-site ferrous ion show that FIH1 undergoes a significant conformational stabilization with a decrease in motional amplitude upon binding of the substrate or the inhibitors. Two loop structures around the active-site reveal a high flexibility in the resting form of FIH1 with the high B-factor values. These high-amplitude motions of the flexible loops are found to be weakened significantly in the presence of the substrate or a weak inhibitor (NOG), and damped out upon binding of a potent and selective inhibitor (NODP) in the active site. A characteristic feature that discriminates the coordination structures of the active-site ferrous ion in complex with 2OG and NOG in solution from those in the X-ray crystal structures lies in the presence of a structural water molecule from bulk solvent at the sixth coordination position, which leads to the formation of a stable octahedral coordination geometry. However, the approach of such a structural water molecule to the active-site ferrous ion is prohibited in the FIH1–NODP complex, which should be attributed to the formation of hydrophobic contacts between the phenyl ring of the inhibitor and the side chains of Tyr102, Leu186, and Trp296 at the entrance of the active site. This indicates that the d-enantiomeric side-chain phenyl group of NODP should play an essential role in potent and selective inhibition of FIH1.

Investigating the dependence of the hypoxia-inducible factor hydroxylases (factor inhibiting HIF and prolyl hydroxylase domain 2) on ascorbate and other reducing agents

The HIF (hypoxia-inducible factor) hydroxylases [PHDs or EGLNs (prolyl hydroxylases), which in humans are PHD isoforms 1-3, and FIH (factor inhibiting HIF)] regulate HIF levels and activity. These enzymes are Fe(II)/2-oxoglutarate-dependent oxygenases, many of which are stimulated by ascorbate. We have investigated the ascorbate dependence of PHD2-catalysed hydroxylation of two prolyl hydroxylation sites in human HIF-1alpha, and of FIH-catalysed hydroxylation of asparaginyl hydroxylation sites in HIF-1alpha and in a consensus ankyrin repeat domain peptide. The initial rate and extent of hydroxylation was increased in the presence of ascorbate for each of these reactions. When ascorbate was replaced with structural analogues, the results revealed that the ascorbate side chain was not important in its contribution to HIF hydroxylase catalysis, whereas modifications to the ene-diol portion of the molecule negated the ability to promote hydroxylation. We investigated whether alternative reducing agents (glutathione and dithiothreitol) could be used to promote HIF hydroxylase activity, and found partial stimulation of hydroxylation in an apparently enzyme- and substrate-specific manner. The results raise the possibility of developing reducing agents targeted to specific HIF hydroxylase-catalysed reactions.

MiR-31 Ablates Expression of the HIF Regulatory Factor FIH to Activate the HIF Pathway in Head and Neck Carcinoma

MicroRNAs (miRNA) are endogenously expressed noncoding RNAs with important biological and pathological functions that are yet to be fully defined. This study investigated alterations in miRNA expression in head and neck squamous cell carcinoma (HNSCC), the incidence of which is rising throughout the world. Initial screening and subsequent analysis identified a panel of aberrantly expressed miRNAs in HNSCC tissues, with miR-31 among the most markedly upregulated. Ectopic expression of miR-31 increased the oncogenic potential of HNSCC cells under normoxic conditions in cell culture or tumor xenografts. Conversely, blocking miR-31 expression reduced the growth of tumor xenografts. The in silico analysis suggested that miR-31 may target the 3' untranslated region (UTR) of factor-inhibiting hypoxia-inducible factor (FIH), a hypoxia-inducible factor (HIF) regulatory factor that inhibits the ability of HIF to act as a transcriptional regulator under normoxic conditions. In support of this likelihood, miR-31 expression repressed FIH expression and mutations within the predictive miR-31 target site in the FIH 3' UTR abrogated FIH repression. Furthermore, miR-31 expression increased HIF transactivation activity. We found that FIH suppressed oncogenic phenotypes under normoxic conditions and that this activity was abrogated by functional mutations. Lastly, increased miR-31 expression was correlated with decreased levels of FIH in tumor tissues. Our findings suggest that miR-31 contributes to the development of HNSCC by impeding FIH to activate HIF under normoxic conditions.

FoxO Proteins Mediate Hypoxic Induction of Connective Tissue Growth Factor in Endothelial Cells

Hypoxia, a driving force in neovascularization, promotes alterations in gene expression mediated by hypoxia-inducible factor (HIF)-1alpha. Connective tissue growth factor (CTGF, CCN2) is a modulator of endothelial cell growth and migration, but its regulation by hypoxia is poorly understood. Therefore, we analyzed signaling pathways involved in the regulation of CTGF by hypoxia in endothelial cells. Exposure to low oxygen tension or treatment with the hypoxia-mimetic dimethyloxalyl glycine (DMOG) stabilized HIF-1alpha and up-regulated CTGF in human umbilical vein endothelial cells and in a murine microvascular endothelial cell line. Induction of CTGF correlated with a HIF-dependent increase in protein and mRNA levels, and nuclear accumulation of the transcription factor FoxO3a. By contrast, gene expression and cellular localization of FoxO1 were not significantly altered by hypoxia. Expression of CTGF was strongly reduced by siRNA silencing of FoxO1 or FoxO3a. Furthermore, nuclear exclusion of FoxO1/3a transcription factors by inhibition of serine/threonine protein phosphatases by okadaic acid inhibited CTGF expression, providing evidence for both FoxO proteins as regulators of CTGF expression. The DMOG-stimulated induction of CTGF was further increased when endothelial cells were co-incubated with transforming growth factor-beta, an activator of Smad signaling. Activation of RhoA-Rho kinase signaling by the microtubule-disrupting drug combretastatin A4 also enhanced the DMOG-induced CTGF expression, thus placing CTGF induction by hypoxia in a network of interacting signaling pathways. Our findings provide evidence that FoxO1, hypoxia-stimulated expression of FoxO3a and its nuclear accumulation are required for the induction of CTGF by hypoxia in endothelial cells.

Intermittent Hypoxia: Potential Factor of Resistance to Endocrine Therapy.

Abstract Background: It has been reported that intermittent hypoxia, which is likely more prevalent than acute hypoxia in breast cancers, can cause persistent depression of estrogen receptor alpha (ER-α) expression in breast cell lines. However, it remains suspension whether intermittent hypoxia may be associated with resistance to endocrine therapy.Material and methods: The role of intermittent hypoxia in resistance to endocrine therapy was investigated in ER-α positive breast cell lines and animal models.Results: We observed that intermittent hypoxia promoted cancer cell proliferation in vitro and in vivo compared with normoxia. At both mRNA and protein levels, intermittent hypoxia induced down-regulation of ER-α and factor inhibiting HIF-1 (FIH) as well as up-regulation of hypoxia inducible factor-1 alpha (HIF-1α) and carbonic anhydrase IX (CA-IX). ER-α positive breast cancer cells under normoxic condition were capable of an intact response to ICI 182,780 (Tocris), whereas the inhibitory effect of ICI 182,780 (Tocris) was significantly reduced in those after intermittent hypoxia. Such effect was further validated with fulvestrant (Astrazeneca) in the animal models. To clarify whether intermittent hypoxia was associated with resistance to endocrine therapy, the expression of HIF-1α, a critical regulator of hypoxia-related pathway, was blocked by a reformed type of small interfering RNA (siRNA), stealth RNAi (Invitrogen), which can reduce the cytotoxic interferon response unlike conventional siRNA. Interestingly, knock-down of HIF-1α did result in the restoration of not only the ER-α expression but also the response to endocrine therapy, which was reproducible with administration of Bortezomib, a proteasome inhibitor of HIF-1α.Discussion: These data provide functional evidence that intermittent hypoxia may confer resistance to endocrine therapy in breast cancers through the crosstalk between HIF-1α and ER-α signaling, which holds a promise to overcome endocrine resistance. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5148.

Signalling Cross Talk of the HIF System: Involvement of the FIH Protein

Cellular and systemic oxygen homeostasis is regulated by an oxygen-sensitive signalling pathway centred on a transcription factor known as Hypoxia Inducible Factor (HIF). Regulation of HIF activity and protein stability is mediated by a family of hydroxylases that act as oxygen sensors due to the dependence of the hydroxylation reaction on oxygen. The transcriptional activity of HIF is at least in part determined by asparaginyl hydroxylation by Factor Inhibiting HIF (FIH) of a C-terminal residue that regulates co-activator recruitment. The activity of FIH on HIF is limiting; emerging data suggest this may be due to competition from a large family of alternative FIH substrates that act as a 'sink' for FIH activity. These alternative substrates are targeted for hydroxylation at conserved Asn residues within a protein interaction domain known as the Ankyrin Repeat Domain (ARD). Many ARD-containing proteins bind to FIH more tightly than does HIF. Furthermore, ARD proteins are common within the proteome and in some cases are highly abundant. Since ARD substrates bind to FIH in a similar manner to HIF it is thought that these properties of the ARD family lead to competitive inhibition of FIH-dependent HIF hydroxylation. We summarise the current literature here and discuss the possible role of cross-talk between the FIH, HIF and ARD systems in fine tuning hypoxia responses.

FIH‐Dependent Asparaginyl Hydroxylation of Ankyrin Repeat Domain‐Containing Proteins

Studies on hypoxia-sensitive pathways have identified a series of Fe(II)-dependent dioxygenases that regulate hypoxia-inducible factor (HIF) by prolyl and asparaginyl hydroxylation. The asparaginyl hydroxylase factor inhibiting HIF (FIH) targets a conserved asparaginyl residue in the C-terminal transactivation domain of HIF-alpha. This modification suppresses HIF transcriptional activity by inhibiting co-activator recruitment. Recent work has demonstrated that FIH targets an alternative class of substrate. Proteins containing a common interaction motif known as the ankyrin repeat domain (ARD) have been shown to be efficiently hydroxylated by FIH. This review aims to summarize what is currently known regarding ARD hydroxylation, including the kinetics and determinants of FIH-mediated ARD hydroxylation, the structural and functional consequences of ARD hydroxylation, and the potential for cross-talk between ARD proteins and HIF signaling.

Structure-based virtual screening approach to the discovery of novel inhibitors of factor-inhibiting HIF-1: Identification of new chelating groups for the active-site ferrous ion

The inhibitors of factor-inhibiting HIF-1 (FIH1) have been shown to be useful as therapeutics for the treatment of anemia. We have been able to identify eight novel FIH1 inhibitors with IC 50 values ranging from 30 to 80 μM by means of the virtual screening with docking simulations under consideration of the effects of ligand solvation in the scoring function. The newly identified inhibitors are structurally diverse and have various chelating groups for the active-site ferrous ion including sulfonamide, carboxylate, N-benzo[1,2,5]oxadiazol-4-yl amide, and 2-[1,2,4]triazolo[3,4- b]][1,3,4]thiadiazol-3-yl-quinoline moieties. Each of these four structural classes has not been reported as FIH1 inhibitor, and therefore can be considered for further development by structure–activity relationship or de novo design methods. The interactions with the amino acid residues responsible for the stabilizations of the inhibitors in the active site are addressed in detail.

Nutlin-3, an Hdm2 antagonist, inhibits tumor adaptation to hypoxia by stimulating the FIH-mediated inactivation of HIF-1α

The interplay among hypoxia-inducible factor 1-alpha (HIF-1alpha), p53 and human orthologue of murine double minute 2 (Hdm2) has been introduced as a key event in tumor promotion and angiogenesis. Recently, nutlin-3, a small-molecule antagonist of Hdm2, was demonstrated to inhibit the HIF-1-mediated vascular endothelial growth factor production and tumor angiogenesis. Yet, the mechanism by which nutlin-3 inhibits HIF-1 is an open question. We here addressed the mode-of-action of nutlin-3 with respect to the HIF-1alpha-p53-Hdm2 interplay. The effect of nutlin-3 on HIF-1alpha function was examined by reporter analyses, immunoprecipitation and immunoblotting. Nutlin-3 downregulated HIF-1alpha, which occurred p53-dependently but von Hippel-Lindau-independently. On the contrary, nutlin-3 blunted the hypoxic induction of vascular endothelial growth factor by inactivating HIF-1 even in p53-null cells. The C-terminal transactivation domain (CAD) of HIF-1alpha was inactivated by nutlin-3, and furthermore, the factor-inhibiting hypoxia-inducible factor (FIH) hydroxylation of Asn803 was required for the nutlin-3 action. In terms of protein interactions, Hdm2 competed with FIH in CAD binding and inhibited the Asn803 hydroxylation both in vivo and in vitro, which facilitated p300 recruitment. Moreover, nutlin-3 reinforced the FIH binding and Ans803 hydroxylation by inhibiting Hdm2. In conclusion, Hdm2 functionally activates HIF-1 by inhibiting the FIH interaction with CAD, and the Hdm2 inhibition by nutlin-3 results in HIF-1 inactivation and vascular endothelial growth factor suppression. The interplays among HIF-1alpha, Hdm2, FIH and p300 could be potential targets for treating tumors overexpressing HIF-1alpha.

Analysis of the role of the HIF hydroxylase family members in erythropoiesis

HIF (hypoxia-inducible factor) hydroxylases have been implicated in EPO (erythropoietin) production and erythropoiesis. Here we examined the role of each of the three EGLN family members and the HIF asparaginyl hydroxylase FIH (factor inhibiting HIF) in EPO production. We examined the effect of inhibiting individual as well as combinations of HIF hydroxylases with RNAi. We found that inhibition of EGLN1 (egl nine homolog 1) as well as other members of the EGLN family (EGLN2 and EGLN3) led to accumulative EPO production in vitro. We then knocked down EGNL1 in vivo by injecting one-cell murine zygotes with lentivirus-containing RNAi. Progeny with demonstrated EGLN1 inhibition had elevated EPO production and erythropoiesis in vivo. Among all the in vitro and in vivo studies, no or minimal VEGF (vascular endothelial growth factor) mRNA or protein stimulation resulted from inhibition of EGLN1.

Asparaginyl β-Hydroxylation of Proteins Containing Ankyrin Repeat Domains Influences Their Stability and Function

Recent reports have provided evidence that the β-hydroxylation of conserved asparaginyl residues in ankyrin repeat domain (ARD) proteins is a common posttranslational modification in animal cells. Here, nuclear magnetic resonance (NMR) and other biophysical techniques are used to study the effect of asparaginyl β-hydroxylation on the structure and stability of ‘consensus’ ARD proteins. The NMR analyses support previous work suggesting that a single β-hydroxylation of asparagine can stabilize the stereotypical ARD fold. A second asparaginyl β-hydroxylation causes further stabilization. In combination with mutation studies, the biophysical analyses reveal that the stabilizing effect of β-hydroxylation is, in part, mediated by a hydrogen bond between the asparaginyl β-hydroxyl group and the side chain of a conserved aspartyl residue, two residues to the N-terminal side of the target asparagine. Removal of this hydrogen bond resulted in reduced stabilization by hydroxylation. Formation of the same hydrogen bond is also shown to be a factor in inhibiting binding of hydroxylated ARDs to factor-inhibiting hypoxia-inducible factor (FIH). The effects of hydroxylation appear to be predominantly localized to the target asparagine and proximal residues, at least in the consensus ARD protein. The results reveal that thermodynamic stability is a factor in determining whether a particular ARD protein is an FIH substrate; a consensus ARD protein with three ankyrin repeats is an FIH substrate, while more stable consensus ARD proteins, with four or five ankyrin repeats, are not. However, NMR studies reveal that the consensus protein with four ankyrin repeats is still able to bind to FIH, suggesting that FIH may interact in cells with natural ankyrin repeats without resulting hydroxylation. Overall, the work provides novel biophysical insights into the mechanism by which asparaginyl β-hydroxylation stabilizes the ARD proteins and reduces their binding to FIH.