Hypoxia inducible factor 1α (HIF1α) is the mammalian transcriptional factor that controls metabolism, survival, and innate immunity in response to inflammation and low oxygen. Previous work established that generation of hypoxic microenvironments occurs within the lung during infection with the human fungal pathogen Aspergillus fumigatus. Here we demonstrate that A. fumigatus stabilizes HIF1α protein early after pulmonary challenge that is inhibited by treatment of mice with the steroid triamcinolone. Utilizing myeloid deficient HIF1α mice, we observed that HIF1α is required for survival and fungal clearance early following pulmonary challenge with A. fumigatus. Unlike previously reported research with bacterial pathogens, HIF1α deficient neutrophils and macrophages were surprisingly not defective in fungal conidial killing. The increase in susceptibility of the myeloid deficient HIF1α mice to A. fumigatus was in part due to decreased early production of the chemokine CXCL1 (KC) and increased neutrophil apoptosis at the site of infection, resulting in decreased neutrophil numbers in the lung. Addition of recombinant CXCL1 restored neutrophil survival and numbers, murine survival, and fungal clearance. These results suggest that there are unique HIF1α mediated mechanisms employed by the host for protection and defense against fungal pathogen growth and invasion in the lung. Additionally, this work supports the strategy of exploring HIF1α as a therapeutic target in specific immunosuppressed populations with fungal infections.
Due to the limited treatment options and severity of invasive fungal infections, a better understanding of fungal-host interactions is needed for the development of new therapies. Recent studies have implicated a role for hypoxia inducible factor 1-alpha (HIF1α) in the regulation of inflammation and host defense responses to microbial pathogens. In this study, we discover that HIF1α is required for protection and murine survival to Aspergillus fumigatus pulmonary challenge. First, we observed that nuclear HIF1α protein levels are reduced in the murine corticosteroid immunosuppressed model of invasive pulmonary aspergillosis, suggesting its involvement in disease outcome. We then tested the hypothesis that HIF1α is required by innate immune effector cells to control/prevent A. fumigatus growth and invasion. Surprisingly, we observed that the role of myeloid HIF1α is not to mediate innate effector cell A. fumigatus killing directly, but rather to induce and maintain a protective immune response that ensures proper effector cell recruitment and survival at the site of infection. These findings provide a better understanding of host mechanisms involved in thwarting fungal pathogenesis, have implications for host susceptibility, and reveal the potential for novel treatment strategies involving HIF1α mediated signaling in the lung in immune suppressed patients.
Solid tumors often exhibit simultaneously inflammatory and hypoxic microenvironments. The ‘signal transducer and activator of transcription-3’ (STAT3)-mediated inflammatory response and the hypoxia-inducible factor (HIF)-mediated hypoxia response have been independently shown to promote tumorigenesis through the activation of HIF or STAT3 target genes and to be indicative of a poor prognosis in a variety of tumors. We report here for the first time that STAT3 is involved in the HIF1, but not HIF2-mediated hypoxic transcriptional response. We show that inhibiting STAT3 activity in MDA-MB-231 and RCC4 cells by a STAT3 inhibitor or STAT3 small interfering RNA significantly reduces the levels of HIF1, but not HIF2 target genes in spite of normal levels of hypoxia-inducible transcription factor 1α (HIF1α) and HIF2α protein. Mechanistically, STAT3 activates HIF1 target genes by binding to HIF1 target gene promoters, interacting with HIF1α protein and recruiting coactivators CREB binding protein (CBP) and p300, and RNA polymerase II (Pol II) to form enhanceosome complexes that contain HIF1α, STAT3, CBP, p300 and RNA Pol II on HIF1 target gene promoters. Functionally, the effect of STAT3 knockdown on proliferation, motility and clonogenic survival of tumor cells in vitro is phenocopied by HIF1α knockdown in hypoxic cells, whereas STAT3 knockdown in normoxic cells also reduces cell proliferation, motility and clonogenic survival. This indicates that STAT3 works with HIF1 to activate HIF1 target genes and to drive HIF1-depedent tumorigenesis under hypoxic conditions, but also has HIF-independent activity in normoxic and hypoxic cells. Identifying the role of STAT3 in the hypoxia response provides further data supporting the effectiveness of STAT3 inhibitors in solid tumor treatment owing to their usefulness in inhibiting both the STAT3 and HIF1 pro-tumorigenic signaling pathways in some cancer types.
cotranscriptional activation; HIF; hypoxia; STAT3; transcription
Hypoxia-inducible factor (HIF) is a nuclear transcription factor that responds to environmental and pathological hypoxia to induce metabolic adaptation, vascular growth, and cell survival. Here we found that HIF subunits and HIF2α in particular were normally expressed in the mediobasal hypothalamus of mice. Hypothalamic HIF was up-regulated by glucose to mediate the feeding control of hypothalamic glucose sensing. Two underlying molecular pathways were identified, including suppression of PHDs by glucose metabolites to prevent HIF2α degradation and the recruitment of AMPK and mTOR/S6K to regulate HIF2α protein synthesis. HIF activation was found to directly control the transcription of POMC gene. Genetic approach was then employed to develop conditional knockout mice with HIF inhibition in POMC neurons, revealing that HIF loss-of-function in POMC neurons impaired hypothalamic glucose sensing and caused energy imbalance to promote obesity development. The metabolic effects of HIF in hypothalamic POMC neurons were independent of leptin signaling or pituitary ACTH pathway. Hypothalamic gene delivery of HIF counteracted overeating and obesity under conditions of nutritional excess. In conclusion, HIF controls hypothalamic POMC gene to direct the central nutrient sensing in regulation of energy and body weight balance.
The hypothalamus in the brain is a master regulator of feeding and body weight. The regulation of it is mediated by the ability of the hypothalamus to sense nutrients (most importantly glucose) and hormones (such as insulin and leptin). While hormone has been extensively studied, we know less about how the hypothalamus can sense nutrients. It is also unclear whether changes in hypothalamic nutrient sensing can influence the development of obesity and related disease, and could therefore be targeted for disease intervention. In this study, we show that a protein termed hypoxia-inducible factor (HIF) is normally present in the hypothalamus and able to respond to glucose. This glucose response leads to the up-regulation of a hypothalamic neuropeptide, POMC, a pivotal molecule that controls feeding and body weight balance. We then developed a mouse model in which HIF is disrupted in hypothalamic cells that express POMC. These mice displayed reduced hypothalamic sensitivity to glucose, resulting in overeating and susceptibility to obesity. Furthermore, we found that delivery of the HIF gene into the hypothalamus has strong anti-obesity effects in mice. We conclude that HIF is a molecular mediator of hypothalamic glucose sensing and can be potentially targeted for obesity therapeutics.
Tuberculosis is a current major world-health problem, exacerbated by the causative pathogen, Mycobacterium tuberculosis (Mtb), becoming increasingly resistant to conventional antibiotic treatment. Mtb is able to counteract the bactericidal mechanisms of leukocytes to survive intracellularly and develop a niche permissive for proliferation and dissemination. Understanding of the pathogenesis of mycobacterial infections such as tuberculosis (TB) remains limited, especially for early infection and for reactivation of latent infection. Signaling via hypoxia inducible factor α (HIF-α) transcription factors has previously been implicated in leukocyte activation and host defence. We have previously shown that hypoxic signaling via stabilization of Hif-1α prolongs the functionality of leukocytes in the innate immune response to injury. We sought to manipulate Hif-α signaling in a well-established Mycobacterium marinum (Mm) zebrafish model of TB to investigate effects on the host's ability to combat mycobacterial infection. Stabilization of host Hif-1α, both pharmacologically and genetically, at early stages of Mm infection was able to reduce the bacterial burden of infected larvae. Increasing Hif-1α signaling enhanced levels of reactive nitrogen species (RNS) in neutrophils prior to infection and was able to reduce larval mycobacterial burden. Conversely, decreasing Hif-2α signaling enhanced RNS levels and reduced bacterial burden, demonstrating that Hif-1α and Hif-2α have opposing effects on host susceptibility to mycobacterial infection. The antimicrobial effect of Hif-1α stabilization, and Hif-2α reduction, were demonstrated to be dependent on inducible nitric oxide synthase (iNOS) signaling at early stages of infection. Our findings indicate that induction of leukocyte iNOS by stabilizing Hif-1α, or reducing Hif-2α, aids the host during early stages of Mm infection. Stabilization of Hif-1α therefore represents a potential target for therapeutic intervention against tuberculosis.
Tuberculosis is a mycobacterial disease that was a major cause of death until the discovery of antibiotics in the mid-twentieth century. However, TB is once again on the rise, with the emergence of strains that are multi-drug resistant. Mycobacteria are specialists in evading immune cell killing and use host immune cells as a niche in which they can proliferate and survive latently, until subsequent re-activation and spreading causing life-threatening disease. Pharmaceutical reprogramming of the immune system to kill intracellular mycobacteria would represent a therapeutic strategy, effective against currently untreatable strains and less susceptible to drug resistance. Here we use an in vivo zebrafish model of TB to show that manipulation of the host genetic pathway responsible for detecting low oxygen levels (hypoxia) causes a decrease in mycobacterial infection. This antimicrobial effect was due to a priming of immune cells with increased levels of nitric oxide, a molecule that is used by immune cells to kill bacteria. Here we show in vivo manipulation of a host-signaling pathway aids the host in combatting mycobacteria infection, identifying hypoxic signaling as a potential target for future therapeutics against TB.
The hypoxic response is an ancient stress response triggered by low ambient oxygen (O2)1. It is controlled by hypoxia inducible transcription factor-1 (HIF-1), whose α subunit is rapidly degraded under normoxic conditions but stabilized when O2-dependent prolyl hydroxylases (PHDs) that target its O2-dependent degradation domain (ODD) are inhibited2–4. Thus the amount of HIF-1α, which controls many genes involved in energy metabolism and angiogenesis is regulated post-translationally. Another ancient stress response is the innate immune response, regulated by several transcription factors, among which NF-κB plays a central role5, 6. NF-κB activation is controlled by IκB kinases (IKK), mainly IKKβ, which are required for phosphorylation-induced degradation of IκB inhibitors in response to infection and inflammation6. Recently, IKKβ was found to be activated in hypoxic cell cultures when PHDs that suppress its activation are inhibited7. However, defining the relationship between NF-κB and HIF-1α has proven elusive. Using in vitro systems, it was reported that HIF-1α activates NF-κB8, that NF-κB controls HIF-1α transcription9 and that activation of HIF-1α may be concurrent to inhibition of NF-κB10. We used mice lacking IKKβ in different cell types to demonstrate that NF-κB is a critical transcriptional activator of HIF-1α in macrophages responding to bacterial infection and in liver and brain of hypoxic animals. IKKβ deficiency results in defective induction of various HIF-1α target genes including vascular endothelial growth factor (VEGF) and elevated astrogliosis in hypoxic mice. Hence, IKKβ provides an important physiological link between the hypoxic response and innate immunity/inflammation, two ancient stress response systems.
While the functions of hypoxia-inducible factor 1α (HIF1α)/aryl hydrocarbon receptor nuclear translocator (ARNT) and HIF2α/ARNT (HIF2) proteins in activating hypoxia-inducible genes are well established, the role of other transcription factors in the hypoxic transcriptional response is less clear. We report here for the first time that the basic helix-loop-helix-leucine-zip transcription factor upstream stimulatory factor 2 (USF2) is required for the hypoxic transcriptional response, specifically, for hypoxic activation of HIF2 target genes. We show that inhibiting USF2 activity greatly reduces hypoxic induction of HIF2 target genes in cell lines that have USF2 activity, while inducing USF2 activity in cells lacking USF2 activity restores hypoxic induction of HIF2 target genes. Mechanistically, USF2 activates HIF2 target genes by binding to HIF2 target gene promoters, interacting with HIF2α protein, and recruiting coactivators CBP and p300 to form enhanceosome complexes that contain HIF2α, USF2, CBP, p300, and RNA polymerase II on HIF2 target gene promoters. Functionally, the effect of USF2 knockdown on proliferation, motility, and clonogenic survival of HIF2-dependent tumor cells in vitro is phenocopied by HIF2α knockdown, indicating that USF2 works with HIF2 to activate HIF2 target genes and to drive HIF2-depedent tumorigenesis.
Chronic alcohol causes hepatic steatosis and liver hypoxia. Hypoxia-regulated Hypoxia-inducible factor 1-α, (HIF1α) may regulate liporegulatory genes but the relationship of HIF1 to steatosis remains unknown. We investigated HIF1α in alcohol-induced hepatic lipid accumulation. Alcohol administration resulted in steatosis, increased liver triglyceride levels and serum ALT suggesting liver injury in WT mice. There was increased hepatic HIF1α mRNA, protein and DNA-binding activity in alcohol-fed mice compared to controls. Mice engineered with hepatocyte-specific HIF1 activation (HIF1dPA) had increased HIF1α mRNA, protein, and DNA-binding activity, and alcohol feeding in HIF1dPA mice increased hepatomegaly and hepatic triglyceride compared to WT. In contrast, hepatocyte-specific deletion of HIF1α (HIF-1α(Hep-/-), protected mice from alcohol- and LPS-induced liver damage, serum ALT elevation, hepatomegaly and lipid accumulation. HIF-1α(Hep-/-), WT, and HIF1dPA mice had equally suppressed levels of PPARα mRNA after chronic ethanol, while the HIF target, ADRP, was upregulated in WT, but not in HIF-1α(Hep-/-) ethanol fed/LPS challenged mice. The chemokine, MCP-1, was cooperatively induced by alcohol feeding and LPS in WT but not in HIF-1α(Hep-/-) mice. Using Huh7 hepatoma cells in vitro, we found that MCP-1 treatment induced lipid accumulation and increased HIF1α protein expression as well as DNA-binding activity. SiRNA inhibition of HIF1α prevented MCP-1-induced lipid accumulation suggesting a mechanistic role for HIF1α in hepatocyte lipid accumulation.
Alcohol feeding results in lipid accumulation in hepatocytes involving HIF1α activation. The alcohol-induced chemokine, MCP-1, triggers lipid accumulation in hepatocytes via HIF1α activation, suggesting a mechanistic link between inflammation and hepatic steatosis in alcoholic liver disease.
Hypoxia-Inducible Factor-1 alpha; Monocyte-Chemoattractant Protein-1; Steatosis; Alcoholic Liver Disease
Otitis media with effusion (OME) is the commonest cause of hearing loss in children, yet the underlying genetic pathways and mechanisms involved are incompletely understood. Ventilation of the middle ear with tympanostomy tubes is the commonest surgical procedure in children and the best treatment for chronic OME, but the mechanism by which they work remains uncertain. As hypoxia is a common feature of inflamed microenvironments, moderation of hypoxia may be a significant contributory mechanism. We have investigated the occurrence of hypoxia and hypoxia-inducible factor (HIF) mediated responses in Junbo and Jeff mouse mutant models, which develop spontaneous chronic otitis media. We found that Jeff and Junbo mice labeled in vivo with pimonidazole showed cellular hypoxia in inflammatory cells in the bulla lumen, and in Junbo the middle ear mucosa was also hypoxic. The bulla fluid inflammatory cell numbers were greater and the upregulation of inflammatory gene networks were more pronounced in Junbo than Jeff. Hif-1α gene expression was elevated in bulla fluid inflammatory cells, and there was upregulation of its target genes including Vegfa in Junbo and Jeff. We therefore investigated the effects in Junbo of small-molecule inhibitors of VEGFR signaling (PTK787, SU-11248, and BAY 43-9006) and destabilizing HIF by inhibiting its chaperone HSP90 with 17-DMAG. We found that both classes of inhibitor significantly reduced hearing loss and the occurrence of bulla fluid and that VEGFR inhibitors moderated angiogenesis and lymphangiogenesis in the inflamed middle ear mucosa. The effectiveness of HSP90 and VEGFR signaling inhibitors in suppressing OM in the Junbo model implicates HIF–mediated VEGF as playing a pivotal role in OM pathogenesis. Our analysis of the Junbo and Jeff mutants highlights the role of hypoxia and HIF–mediated pathways, and we conclude that targeting molecules in HIF–VEGF signaling pathways has therapeutic potential in the treatment of chronic OM.
Otitis media with effusion (OME) is the commonest cause of hearing loss in children, and treatment using grommets remains the commonest surgical procedure in children. Chronic forms of OM are known from human population studies to have a significant genetic component, but little is known of the underlying genes or pathways involved. We have analyzed two chronic OM mouse models, the Junbo and Jeff mutants, and have found that both demonstrate hypoxia and hypoxia-inducible factor (HIF) mediated responses. There is upregulation of inflammatory pathways in the mutant middle ears and in Junbo elevation of cytokines that modulate Hif-1α. Hif-1α levels are raised in the middle ear as well as downstream targets of HIF such as Vegfa. We explored the effects of small-molecule inhibitors of HSP90 and VEGF receptor signaling in the Junbo mutant and found significant reductions in hearing loss, the occurrence of bulla fluid, and moderation of vascular changes in the inflamed middle ear mucosa with the VEGF receptor inhibitors. The study of the Junbo and Jeff mutants demonstrates the role of hypoxia and HIF mediated pathways in OM pathogenesis, and it indicates that targeting the HIF–VEGF pathway may represent a novel approach to therapeutic intervention in chronic OM.
Hypoxia inducible factors HIF1α and HIF2α are important proteins involved in the regulation of the transcription of a variety of genes related to erythropoiesis, glycolysis and angiogenesis. Hypoxic stimulation results in rapid increase of the HIF1α and 2α protein levels, as a consequence of a redox-sensitive stabilization. The HIFαs enter the nucleus, heterodimerize with the HIF1β protein, and bind to DNA at the hypoxia response elements (HREs) of target genes. In this study we evaluated the immunohistochemical expression of these proteins in 108 tissue samples from non-small-cell lung cancer (NSCLC) and in normal lung tissues. Both proteins showed a mixed cytoplasmic/nuclear pattern of expression in cancer cells, tumoural vessels and tumour-infiltrating macrophages, as well as in areas of metaplasia, while normal lung components showed negative or very weak cytoplasmic staining. Positive HIF1α and HIF2α expression was noted in 68/108 (62%) and in 54/108 (50%) of cases respectively. Correlation analysis of HIF2α expression with HIF1α expression showed a significant association (P < 0.0001, r = 0.44). A strong association of the expression of both proteins with the angiogenic factors VEGF (P < 0.004), PD-ECGF (P < 0.003) and bFGF (P < 0.04) was noted. HIF1α correlated with the expression of bek-bFGF receptor expression (P = 0.01), while HIF2α was associated with intense VEGF/KDR-activated vascularization (P = 0.002). HIF2α protein was less frequently expressed in cases with a medium microvessel density (MVD); a high rate of expression was noted in cases with both low and high MVD (P = 0.006). Analysis of overall survival showed that HIF2α expression was related to poor outcome (P = 0.008), even in the group of patients with low MVD (P = 0.009). HIF1α expression was marginally associated with poor prognosis (P = 0.08). In multivariate analysis HIF2α expression was an independent prognostic indicator (P = 0.006, t-ratio 2.7). We conclude that HIF1α and HIF2α overexpression is a common event in NSCLC, which is related to the up-regulation of various angiogenic factors and with poor prognosis. Targeting the HIF pathway may prove of importance in the treatment of NSCLC. © 2001 Cancer Research Campaignhttp://www.bjcancer.com
non-small-cell lung cancer; hypoxia inducible factors; angiogenesis; prognosis
Hypoxic tumor cells overexpressing hypoxia-inducible factor 1alpha (HIF-1α) are generally resistant to chemo/radiotherapy. We have reported that Se-methylselenocysteine (MSC) therapeutically enhances the efficacy and selectivity of irinotecan against human tumor xenografts. The aim of this study was to delineate the mechanism responsible for the observed efficacy targeting on HIF-1α and its transcriptionally regulated genes VEGF and CAIX.
We investigated the mechanism of HIF-1α inhibition by MSC and its critical role in the therapeutic outcome by generating HIF-1α stable knockdown (KD) human head and neck squamous cell carcinoma, FaDu by transfecting HIF-1α short hairpin RNA.
While cytotoxic efficacy in combination with methylselenic acid (MSA) with SN-38 (active metabolites of MSC and irinotecan) could not be confirmed in vitro against normoxic tumor cells, the hypoxic tumor cells were more sensitive to the combination. Reduction in HIF-1α either by MSA or shRNA knockdown resulted in significant increase in cytotoxicity of SN38 in vitro against hypoxic, but not the normoxic tumor cells. Similarly, in vivo, either MSC in combination with irinotecan treatment of parental xenografts or HIF-1α KD tumors treated with irinotecan alone resulted in comparable therapeutic response and increase in the long-term survival of mice bearing FaDu xenografts.
Our results show that HIF-1α is a critical target for MSC and its inhibition was associated with enhanced antitumor activity of irinotecan. Inhibition of HIF-1α appeared to be mediated through stabilization of PHD2, 3 and downregulation of ROS by MSC. Thus, our findings support the development of MSC as a HIF-1α inhibitor in combination chemotherapy.
HIF-1α; Se-methylselenocysteine; Irinotecan; Hypoxic tumor cells; PHD
Background: Hypoxia inducible factor-α (HIF-α) is the main transcription factor activated in low oxygen conditions.
Results: Single cell imaging reveals pulses in nuclear levels of HIF-α.
Conclusion: The transient nature of the HIF-α nuclear accumulation is required to avoid cell death.
Significance: The duration of HIF-α response depends on cellular oxygenation, and can encode information and dictate cell fate.
Intracellular signaling involving hypoxia-inducible factor (HIF) controls the adaptive responses to hypoxia. There is a growing body of evidence demonstrating that intracellular signals encode temporal information. Thus, the dynamics of protein levels, as well as protein quantity and/or localization, impacts on cell fate. We hypothesized that such temporal encoding has a role in HIF signaling and cell fate decisions triggered by hypoxic conditions. Using live cell imaging in a controlled oxygen environment, we observed transient 3-h pulses of HIF-1α and -2α expression under continuous hypoxia. We postulated that the well described prolyl hydroxylase (PHD) oxygen sensors and HIF negative feedback regulators could be the origin of the pulsatile HIF dynamics. We used iterative mathematical modeling and experimental analysis to scrutinize which parameter of the PHD feedback could control HIF timing and we probed for the functional redundancy between the three main PHD proteins. We identified PHD2 as the main PHD responsible for HIF peak duration. We then demonstrated that this has important consequences, because the transient nature of the HIF pulse prevents cell death by avoiding transcription of p53-dependent pro-apoptotic genes. We have further shown the importance of considering HIF dynamics for coupling mathematical models by using a described HIF-p53 mathematical model. Our results indicate that the tight control of HIF transient dynamics has important functional consequences on the cross-talk with key signaling pathways controlling cell survival, which is likely to impact on HIF targeting strategies for hypoxia-associated diseases such as tumor progression and ischemia.
Cell Death; Hypoxia; Hypoxia-inducible Factor; Imaging; Mathematical Modeling; Negative Feedback Loop; p53; Prolyl Hydroxylase
Although increasing evidence supports a link between epidermal growth factor receptor (EGFR) signaling and resistance to apoptosis, the mechanism by which the EGFR signaling pathway inhibits apoptosis is not well understood. In this study, we found that epidermal growth factor (EGF) stimulation increased the level of expression of the inhibitor of apoptosis protein survivin in breast cancer cells but not in normal mammary epithelial cells. We further demonstrated that activation of survivin gene expression is mediated by oxygen-independent hypoxia-inducible factor (HIF)-1α up-regulation in EGF-treated cancer cells. EGFR signaling activated the phosphoinositide 3-kinase/AKT pathway, subsequently increasing the level of HIF-1α under normoxic conditions. HIF-1α then activated survivin gene transcription through direct binding to the survivin promoter. Furthermore, we found that overexpression of HIF-1α small interfering RNA blocks EGF-induced survivin gene up-regulation and increases apoptosis induced by the chemotherapy drug docetaxel. However, transfection of a plasmid expressing HIF-1α gene activates survivin gene expression and reduces the apoptotic response. Our results demonstrate a novel pathway for EGFR signaling-mediated apoptosis resistance in human cancer cells. Although the role of HIF-1α in regulating cell survival under hypoxic conditions has been studied extensively, our results show that normoxic breast cancer cells utilize cross-talk between EGFR signals and HIF-1α to up-regulate the anti-apoptotic survivin gene, providing a strong rationale for the targeting of HIF-1α as a therapeutic approach for both hypoxic and normoxic tumor cells. Understanding key molecular events in EGFR signaling-induced apoptosis resistance should provide new information for the development of novel therapeutic agents targeting EGFR, HIF-1α, and/or survivin.
Evidence suggests that the activation of the transcription factor hypoxia-inducible factor 1α (HIF-1α) may promote cell survival in hypoxic or ischemic brain. To help understand the role of HIF-1α in neonatal hypoxic-ischemic brain injury, mice with conditional neuron-specific inactivation of HIF-1α underwent hypoxia-ischemia (HI). Mice heterozygous for Cre recombinase under the control of the calcium/calmodulin-dependent kinase II promoter were bred with homozygous ‘floxed’ HIF-1α transgenic mice. The resulting litters produced mice with a forebrain predominant neuronal deletion of HIF-1α (HIF-1αΔ/Δ), as well as littermates without the deletion. In order to verify reduction of HIF-1α at postnatal day 7, HIF-1αΔ/Δ and wild-type mice were exposed to a hypoxic stimulus (8% oxygen) or room air for 1 h, followed by immediate collection of brain cortices for determination of HIF-1α expression. Results of Western blotting of mouse cortices exposed to hypoxia stimulus or room air confirmed that HIF-1αΔ/Δ cortex expressed a minimal amount of HIF-1α protein compared to wild-type cortex with the same hypoxic stimulus. Subsequently, pups underwent the Vannucci procedure of HI at postnatal day 7: unilateral ligation of the right common carotid artery followed by 30 min of hypoxia (8% oxygen). Immunofluorescent staining of brains 24 h after HI confirmed a relative lack of HIF-1α in the HIF-1αΔ/Δ cortex compared to the wild type, and that HIF-1α in the wild type is located in neurons. HIF-1α expression was determined in mouse cortex 24 h after HI. Histological analysis for the degree of injury was performed 5 days after HI. HIF-1α protein expression 24 h after HI showed a large increase of HIF-1α in the hypoxic-ischemic cortex of the wild-type compared to the hypoxic only cortex. Histological analysis revealed that HI injury was increased in the neuronally deficient HIF-1αΔ/Δ mouse brain (p < 0.05) and was more severe in the cortex. Genetic reduction of neuronal HIF-1α results in a worsening of injury after neonatal HI, with a region-specific role for HIF-1α in the setting of neonatal brain injury.
Brain injury; Transcription factor; Hypoxia-inducible factor 1α; Neonatal hypoxia-ischemia
Small-molecule inhibition of hypoxia-inducible factor prolyl 4-hydroxylases (HIF-P4Hs) is being explored for the treatment of anemia. Previous studies have suggested that HIF-P4H-2 inhibition may also protect the heart from an ischemic insult. Hif-p4h-2gt/gt mice, which have 76 to 93% knockdown of Hif-p4h-2 mRNA in endothelial cells, fibroblasts, and cardiomyocytes and normoxic stabilization of Hif-α, were subjected to ligation of the left anterior descending coronary artery (LAD). Hif-p4h-2 deficiency resulted in increased survival, better-preserved left ventricle (LV) systolic function, and a smaller infarct size. Surprisingly, a significantly larger area of the LV remained perfused during LAD ligation in Hif-p4h-2gt/gt hearts than in wild-type hearts. However, no difference was observed in collateral vessels, while the size of capillaries, but not their number, was significantly greater in Hif-p4h-2gt/gt hearts than in wild-type hearts. Hif-p4h-2gt/gt mice showed increased cardiac expression of endothelial Hif target genes for Tie-2, apelin, APJ, and endothelial nitric oxide (NO) synthase (eNOS) and increased serum NO concentrations. Remarkably, blockage of Tie-2 signaling was sufficient to normalize cardiac apelin and APJ expression and resulted in reversal of the enlarged-capillary phenotype and ischemic cardioprotection in Hif-p4h-2gt/gt hearts. Activation of the hypoxia response by HIF-P4H-2 inhibition in endothelial cells appears to be a major determinant of ischemic cardioprotection and justifies the exploration of systemic small-molecule HIF-P4H-2 inhibitors for ischemic heart disease.
Hypoxia-inducible factor-1 (HIF-1) is the major hypoxia-regulated transcription factor that regulates cellular responses to low oxygen environments. HIF-1 is composed of two subunits: hypoxia-inducible HIF-1α and constitutively-expressed HIF-1β. During hypoxic conditions, HIF-1α heterodimerizes with HIF-1β and translocates to the nucleus where the HIF-1 complex binds to the hypoxia-response element (HRE) and activates expression of target genes implicated in cell growth and survival. HIF-1α protein expression is elevated in many solid tumors, including those of the cervix and brain, where cells that are the greatest distance from blood vessels, and therefore the most hypoxic, express the highest levels of HIF-1α. Therapeutic blockade of the HIF-1 signaling pathway in cancer cells therefore provides an attractive strategy for development of anticancer drugs. To identify small molecule inhibitors of the HIF-1 pathway, we have developed a cell-based reporter gene assay and screened a large compound library by using a quantitative high-throughput screening (qHTS) approach.
The assay is based upon a β-lactamase reporter under the control of a HRE. We have screened approximate 73,000 compounds by qHTS, with each compound tested over a range of seven to fifteen concentrations. After qHTS we have rapidly identified three novel structural series of HIF-1 pathway Inhibitors. Selected compounds in these series were also confirmed as inhibitors in a HRE β-lactamase reporter gene assay induced by low oxygen and in a VEGF secretion assay. Three of the four selected compounds tested showed significant inhibition of hypoxia-induced HIF-1α accumulation by western blot analysis.
The use of β-lactamase reporter gene assays, in combination with qHTS, enabled the rapid identification and prioritization of inhibitors specific to the hypoxia induced signaling pathway.
We recently demonstrated in a mouse model of glaucoma that endogenous epigenetic mechanisms can be activated by a repetitive hypoxic preconditioning (RHP) stimulus to provide robust retinal ganglion cell (RGC) protection. Although we also provided evidence that RHP prevents or delays the apoptotic demise of the RGC soma, the mechanisms responsible for signaling this epigenetic response, as well as the effectors of the glaucoma-tolerant phenotype at the somatic and axonal levels, remain unidentified. In the present study, we used conditional mutant mice lacking hypoxia-inducible factor-1α (HIF-1α) in RGCs (HIF-1α RGC-knockout [KO] mice) to test the hypothesis that RHP-mediated activation of this transcription factor in these cells protects them from glaucomatous injury.
Adult HIF-1α RGC-KO mice, generated by mating floxed HIF-1α mice with math5-Cre mice, were used. Experimental glaucoma was induced unilaterally in the HIF-1α RGC-KO mice and matched wild-types by elevating the intraocular pressure to 16–20 mmHg for 3 consecutive weeks, secondary to episcleral vein ligation. Mice of each genotype were randomized to either an RHP protocol (six total exposures to systemic hypoxia [11% oxygen], interspersed over a 2-week period, completed 3 days before ligation surgery) or to an untreated group. RGC soma and axon injury was quantified with Neuronal Nuclei (NeuN) immunohistochemistry in retinal flat mounts and SMI32 immunohistochemistry in cross sections of the post-laminar optic nerve, respectively.
HIF-1α RGC-KO mice exhibited normal retinal function and morphology, and crosses of math5-Cre mice with floxed ROSA26 reporter mice confirmed Cre recombinase activity was confined to the RGC axons and soma. Untreated wild-type mice exhibited a 30±2% loss of RGC soma and a 31±3% loss of RGC axons after 3 weeks of intraocular hypertension (both p<0.05 versus fellow eye). The 90% and 81% improvement in soma and axon survival, respectively, observed in the wild-type mice treated with RHP (both p<0.05 versus the glaucoma eye in the untreated mice) was still observed to a near identical extent in the RHP-treated HIF-1α RGC-KO mice. RHP had no effect on the magnitude of intraocular pressure elevation in either the KO or wild-type groups, indicating that protection was realized in both genotypes in the face of ongoing intraocular hypertension.
These findings indicate that the robust, glaucomatous protection of the RGC soma and axons induced by RHP does not require HIF-1α-mediated transcription of survival genes and other adaptive responses within the RGCs themselves. Rather, we infer that RGC survival is augmented secondary to the activation of other hypoxia-sensitive transcription factors in RGCs and/or the action of diffusible HIF-1α target gene proteins released from neighboring retinal cells. Ideally, the involvement of such autocrine- and/or paracrine-based mechanisms would be confirmed in future studies, but distinct components of the integrated, pleiotropic, and multicellular basis of this endogenous epigenetic response may prove difficult to demonstrate experimentally, as we found in the present study.
Macrophage secretion of VEGF in response to the hypoxic tumor microenvironment contributes to tumor growth, angiogenesis, and metastasis. We have recently demonstrated that macrophages stimulated with GM-CSF at low O2 secrete high levels of a soluble form of the VEGF receptor (sVEGFR-1), which neutralizes VEGF and inhibits its biological activity. Using siRNA targeting to deplete HIF-1α or HIF-2α in murine macrophages, we found that macrophage production of sVEGFR-1 in response to low O2 was dependent on HIF-2α, while HIF-1α specifically regulated VEGF production. In our current report, we evaluated the growth of B16F10 malignant melanoma in mice with a monocyte/macrophage-selective deletion of HIF-1α or HIF-2α (HIF-1αflox/flox-or HIF-2αflox/+/LysMcre mice). GM-CSF treatment increased intra-tumoral VEGF and sVEGFR-1 in control mice, an effect that was associated with a decrease in microvessel density. GM-CSF treatment of HIF-1αflox/flox/LysMcre mice induced sVEGFR-1 but not VEGF, resulting in an overall greater reduction in tumor growth and angiogenesis compared to control mice. In addition, real-time PCR for melanoma-specific genes revealed a significantly reduced presence of lung micrometastases in HIF-1αflox/flox/LysMcre mice treated with GM-CSF. Conversely, GM-CSF treatment induced VEGF but not sVEGFR-1 in HIF-2αflox/+/LysMcre mice, and correspondingly, GM-CSF did not decrease tumor growth, angiogenesis, or lung metastasis in these mice. This study reveals opposing roles for the HIFs in the regulation of angiogenesis by tumor-associated macrophages, and suggests that administration of GM-CSF might be an effective means of inducing sVEGFR-1 and inhibiting tumor growth and angiogenesis in patients with melanoma.
Hypoxia plays a major role in the induction of angiogenesis during tumor development. One mechanism by which tumor cells respond to a reduced oxygen level is via the activation of hypoxia-inducible factor-1 (HIF-1). HIF-1 is an oxygen-dependent transcriptional activator that plays crucial roles in the angiogenesis of tumors and mammalian development. HIF-1 consists of a constitutively expressed HIF-1β subunit and the highly regulated HIF-1α subunits. The stability and activity of HIF-1α are regulated by various post-translational modifications, hydroxylation, acetylation, phosphorylation and sumoyaltion. Therefore, HIF-1α interacts with several protein factors including PHD, pVHL, ARD-1, SUMO and p300/CBP. Under normoxia, the HIF-1α subunit is rapidly degraded via the von Hippel-Lindau tumor suppressor gene product (pVHL)-mediated ubiquitin/proteasome pathway. The association of pVHL and HIF-1α under normoxic conditions is triggered by the hydroxylation of prolines and the acetylation of lysine within a polypeptide segment known as the oxygen-dependent degradation (ODD) domain. On the contrary, under the hypoxia condition, the HIF-1α subunit becomes stable and interacts with coactivators such as p300/CBP to modulate its transcriptional activity. Under hypoxic conditions, HIF-1 eventually acts as a master regulator of numerous hypoxia-inducible genes. The target genes of HIF-1 are especially related to angiogenesis, cell proliferation and survival, and to glucose and iron metabolism. Moreover, it was reported that the activation of HIF-1α is closely associated with a variety of tumors and oncogenic pathways. Hence, the blocking of HIF-1α itself or the blocking of HIF-1α interacting proteins inhibits tumor growth. Based on these findings, HIF-1 can be a prime target for anticancer therapies. Therefore, this review summarizes the molecular mechanism of HIF-1α stability, the biological functions of HIF-1 and its potential applications for cancer therapies.
ARD1; Angiogenesis; Anticancer therapy; Cell proliferation/survival; Glucose metabolism; HIF-1; Iron metabolism; PHD; SUMO; pVHL; p300/CBP; Transcription factor
Glioblastomas are lethal cancers characterized by florid angiogenesis promoted in part by glioma stem cells (GSCs). As hypoxia regulates angiogenesis, we examined hypoxic responses in GSCs. We now demonstrate that hypoxia-inducible factor HIF2α and multiple HIF-regulated genes are preferentially expressed in GSCs in comparison to nonstem tumor cells and normal neural progenitors. In tumor specimens, HIF2α co-localizes with cancer stem cell markers. Targeting HIFs in GSCs inhibits self-renewal, proliferation and survival in vitro, and attenuates tumor initiation potential of GSCs in vivo. Analysis of a molecular database reveals that HIF2A expression correlates with poor glioma patient survival. Our results demonstrate that GSCs differentially respond to hypoxia with distinct HIF induction patterns and HIF2α may represent a promising target for anti-glioblastoma therapies.
Recent evidence supports the presence of cancer stem cell populations that contribute to tumor progression through preferential resistance to radiation and chemotherapy, and promotion of tumor angiogenesis, invasion, and metastasis. Therefore, the elucidation of molecular regulators of cancer stem cells may translate into improved anti-neoplastic therapies. Our work demonstrates that cancer stem cells derived from glioblastomas differentially respond to hypoxia with a distinct induction of HIF2α. We find that HIFs are critical to cancer stem cell maintenance and angiogenic drive, and that expression of HIF2α is significantly associated with poor glioma patient survival. These data further suggest that anti-angiogenic therapies can be designed to target cancer stem cell specific molecules involved in neoangiogenesis, including HIF2α and its regulated factors.
The transcription factor hypoxia-inducible factor 1 (HIF-1) has recently emerged to be a crucial regulator of the immune response following pathogen perception, including the response to the important human pathogen Pseudomonas aeruginosa. However, as mechanisms involved in HIF-1 activation by bacterial pathogens are not fully characterized, understanding how bacteria and bacterial compounds impact on HIF-1α stabilization remains a major challenge. In this context, we have focused on the effect of secreted factors of P. aeruginosa on HIF-1 regulation. Surprisingly, we found that P. aeruginosa cell-free supernatant significantly repressed HIF-1α protein levels. Further characterization revealed that HIF-1α downregulation was dependent on a subset of key secreted factors involved in P. aeruginosa pathogenesis, the 2-alkyl-4-quinolone (AQ) quorum sensing (QS) signaling molecules, and in particular the pseudomonas quinolone signal (PQS). Under hypoxic conditions, the AQ-dependent downregulation of HIF-1α was linked to the suppressed induction of the important HIF-1 target gene hexokinase II. Furthermore, we demonstrated that AQ molecules directly target HIF-1α protein degradation through the 26S-proteasome proteolytic pathway but independently of the prolyl hydroxylase domain (PHD). In conclusion, this is the first report showing that bacterial molecules can repress HIF-1α protein levels. Manipulation of HIF-1 signaling by P. aeruginosa AQs could have major consequences for the host response to infection and may facilitate the infective properties of this pathogen.
Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric transcription factor containing an inducibly expressed HIF-1α subunit and a constititutively expressed HIF-1β subunit. Under hypoxic conditions, the HIF-1α subunit accumulates due to a decrease in the rate of proteolytic degradation, and the resulting HIF-1α-HIF-1β heterodimers undergo posttranslational modifications that promote transactivation. Recent studies suggest that amplified signaling through phosphoinositide 3-kinase, and its downstream target, mTOR, enhances HIF-1-dependent gene expression in certain cell types. In the present study, we have explored further the linkage between mTOR and HIF-1 in PC-3 prostate cancer cells treated with hypoxia or the hypoxia mimetic agent, CoCl2. Pretreatment of PC-3 cells with the mTOR inhibitor, rapamycin, inhibited both the accumulation of HIF-1α and HIF-1-dependent transcription induced by hypoxia or CoCl2. Transfection of these cells with wild-type mTOR enhanced HIF-1 activation by hypoxia or CoCl2, while expression of a rapamycin-resistant mTOR mutant rendered both HIF-1α stabilization and HIF-1 transactivating function refractory to inhibition by rapamycin. Studies with GAL4-HIF-1α fusion proteins pinpointed the oxygen-dependent degradation domain as a critical target for the rapamycin-sensitive, mTOR-dependent signaling pathway leading to HIF-1α stabilization by CoCl2. These studies position mTOR as an upstream activator of HIF-1 function in cancer cells and suggest that the antitumor activity of rapamycin is mediated, in part, through the inhibition of cellular responses to hypoxic stress.
Sphingosine-1-phosphate (S1P) is a bioactive lipid, which regulates several cancer-related processes including migration and angiogenesis. We have previously shown S1P to induce migration of follicular ML-1 thyroid cancer cells. Hypoxia-induced factor-1 (HIF-1) is an oxygen-sensitive transcription factor, which adapts cells to hypoxic conditions through increased survival, motility and angiogenesis. Due to these properties and its increased expression in response to intratumoral hypoxia, HIF-1 is considered a significant regulator of tumor biology. We found S1P to increase expression of the regulatory HIF-1α subunit in normoxic ML-1 cells. S1P also increased HIF-1 activity and expression of HIF-1 target genes. Importantly, inhibition or knockdown of HIF-1α attenuated the S1P-induced migration of ML-1 cells. S1P-induced HIF-1α expression was mediated by S1P receptor 3 (S1P3), Gi proteins and their downstream effectors MEK, PI3K, mTOR and PKCβI. Half-life measurements with cycloheximide indicated that S1P treatment stabilized the HIF-1α protein. On the other hand, S1P activated translational regulators eIF-4E and p70S6K, which are known to control HIF-1α synthesis. In conclusion, we have identified S1P as a non-hypoxic regulator of HIF-1 activity in thyroid cancer cells, studied the signaling involved in S1P-induced HIF-1α expression and shown S1P-induced migration to be mediated by HIF-1.
Hepcidin is a major regulator of iron metabolism and plays a key role in anemia of chronic disease, reducing intestinal iron uptake and release from body iron stores. Hypoxia and chemical stabilizers of the hypoxia-inducible transcription factor (HIF) have been shown to suppress hepcidin expression. We therefore investigated the role of HIF in hepcidin regulation.
Hepcidin mRNA was down-regulated in hepatoma cells by chemical HIF stabilizers and iron chelators, respectively. In contrast, the response to hypoxia was variable. The decrease in hepcidin mRNA was not reversed by HIF-1α or HIF-2α knock-down or by depletion of the HIF and iron regulatory protein (IRP) target transferrin receptor 1 (TfR1). However, the response of hepcidin to hypoxia and chemical HIF inducers paralleled the regulation of transferrin receptor 2 (TfR2), one of the genes critical to hepcidin expression. Hepcidin expression was also markedly and rapidly decreased by serum deprivation, independent of transferrin-bound iron, and by the phosphatidylinositol 3 (PI3) kinase inhibitor LY294002, indicating that growth factors are required for hepcidin expression in vitro. Hepcidin promoter constructs mirrored the response of mRNA levels to interleukin-6 and bone morphogenetic proteins, but not consistently to hypoxia or HIF stabilizers, and deletion of the putative HIF binding motifs did not alter the response to different hypoxic stimuli. In mice exposed to carbon monoxide, hypoxia or the chemical HIF inducer N-oxalylglycine, liver hepcidin 1 mRNA was elevated rather than decreased.
Taken together, these data indicate that hepcidin is neither a direct target of HIF, nor indirectly regulated by HIF through induction of TfR1 expression. Hepcidin mRNA expression in vitro is highly sensitive to the presence of serum factors and PI3 kinase inhibition and parallels TfR2 expression.
In C. elegans, hydrogen sulfide (H2S) exposure results in Hif-1 stabilization. hif-1 is required for survival in H2S and constitutive HIF-1 stabilization confers resistance to H2S. H2S-induced HIF-1 reporter activity appears to be independent of VHL-1, whereas VHL-1 is required for hypoxic regulation of HIF-1 reporter activity.
Rapid alteration of gene expression in response to environmental changes is essential for normal development and behavior. The transcription factor hypoxia-inducible factor (HIF)-1 is well known to respond to alterations in oxygen availability. In nature, low oxygen environments are often found to contain high levels of hydrogen sulfide (H2S). Here, we show that Caenorhabditis elegans can have mutually exclusive responses to H2S and hypoxia, both involving HIF-1. Specifically, H2S results in HIF-1 activity throughout the hypodermis, whereas hypoxia causes HIF-1 activity in the gut as judged by a reporter for HIF-1 activity. C. elegans require hif-1 to survive in room air containing trace amounts of H2S. Exposure to H2S results in HIF-1 nuclear localization and transcription of HIF-1 targets. The effects of H2S on HIF-1 reporter activity are independent of von Hippel–Lindau tumor suppressor (VHL)-1, whereas VHL-1 is required for hypoxic regulation of HIF-1 reporter activity. Because H2S is naturally produced by animal cells, our results suggest that endogenous H2S may influence HIF-1 activity.
Disulfiram is an aldehyde dehydrogenase inhibitor that was used to treat alcoholism and showed anticancer activity, but its anticancer mechanism remains unclear. The aim of this study was to investigate the effects of disulfiram on the hypoxia-inducible factor (HIF)-driven tumor adaptation to hypoxia in vitro.
Hep3B, Huh7 and HepG2 hepatoma cells were incubated under normoxic (20% O2) or hypoxic (1% O2) conditions for 16 h. The expression and activity of HIF-1α and HIF-2α proteins were evaluated using immunoblotting and luciferase reporter assay, respectively. Semi-quantitative RT-PCR was used to analyze HIF-mediated gene expression. Endothelial tubule formation assay was used to evaluate the anti-angiogenic effect.
Hypoxia caused marked expression of HIF-1α and HIF-1α in the 3 hepatoma cell lines, dramatically increased HIF activity and induced the expression of HIF downstream genes (EPO, CA9, VEGF-A and PDK1) in Hep3B cells. HIF-2α expression was positively correlated with the induction of hypoxic genes (CA9, VEGF-A and PDK1). Moreover, hypoxia markedly increased VEGF production and angiogenic potential of Hep3B cells. Disulfiram (0.3 to 2 μmol/L) inhibited hypoxia-induced gene expression and HIF activity in a dose-dependent manner. Disulfiram more effectively suppressed the viability of Hep3B cells under hypoxia, but it did not affect the cell cycle. Overexpression of HIF-2α in Hep3B cells reversed the inhibitory effects of disulfiram on hypoxia-induced gene expression and cell survival under hypoxia.
Disulfiram deregulates the HIF-mediated hypoxic signaling pathway in hepatoma cells, which may contribute to its anticancer effect. Thus, disulfiram could be used to treat solid tumors that grow in a HIF-dependent manner.
disulfiram; hepatoma; hypoxia; HIF-2; VEGF; angiogenesis