Transcription Factor Hypoxia-inducible Factor-1α Research Articles

Design, synthesis, and screening of novel ursolic acid derivatives as potential anti-cancer agents that target the HIF-1α pathway

The transcription factor hypoxia-inducible factor-1α (HIF-1α) plays an important role in tumor angiogenesis, growth, and metastasis and is recognized as an important potential therapeutic target for cancer. Here, we designed and synthesized three novel series of ursolic acid derivatives containing an aminoguanidine moiety and evaluated them as HIF-1α inhibitors and anti-cancer agents using human cancer cell lines. Most of the compounds exhibited significant inhibition of HIF-1α transcriptional activity, as measured using a Hep3B cell-based luciferase reporter assay. Among these compounds, 7b was the most potent inhibitor of HIF-1α expression under hypoxic conditions (IC50 4.0 µM) and did not display significant cytotoxicity against any cell lines tested. The mechanism of action of 7b was investigated, we found that 7b downregulated HIF-1α protein expression, possibly by suppressing its synthesis, reduced production of vascular endothelial growth factor, and inhibited the proliferation of cancer cells.

Targeting the Warburg effect via LDHA inhibition engages ATF4 signaling for cancer cell survival.

Nutrient restriction reprograms cellular signaling and metabolic network to shape cancer phenotype. Lactate dehydrogenase A (LDHA) has a key role in aerobic glycolysis (the Warburg effect) through regeneration of the electron acceptor NAD+ and is widely regarded as a desirable target for cancer therapeutics. However, the mechanisms of cellular response and adaptation to LDHA inhibition remain largely unknown. Here, we show that LDHA activity supports serine and aspartate biosynthesis. Surprisingly, however, LDHA inhibition fails to impact human melanoma cell proliferation, survival, or tumor growth. Reduced intracellular serine and aspartate following LDHA inhibition engage GCN2-ATF4 signaling to initiate an expansive pro-survival response. This includes the upregulation of glutamine transporter SLC1A5 and glutamine uptake, with concomitant build-up of essential amino acids, and mTORC1 activation, to ameliorate the effects of LDHA inhibition. Tumors with low LDHA expression and melanoma patients acquiring resistance to MAPK signaling inhibitors, which target the Warburg effect, exhibit altered metabolic gene expression reminiscent of the ATF4-mediated survival signaling. ATF4-controlled survival mechanisms conferring synthetic vulnerability to the approaches targeting the Warburg effect offer efficacious therapeutic strategies.

High salt intake shifts the mechanisms of flow-induced dilation in the middle cerebral arteries of Sprague-Dawley rats.

The goal of the present study was to examine the effect of 1 wk of high salt (HS) intake and the role of oxidative stress in changing the mechanisms of flow-induced dilation (FID) in isolated pressurized middle cerebral arteries of male Sprague-Dawley rats ( n = 15-16 rats/group). Reduced FID in the HS group was restored by intake of the superoxide scavenger tempol (HS + tempol in vivo group). The nitric oxide (NO) synthase inhibitor Nω-nitro-l-arginine methyl ester, cyclooxygenase inhibitor indomethacin, and selective inhibitor of microsomal cytochrome P-450 epoxidase activity N-(methylsulfonyl)-2-(2-propynyloxy)-benzenehexanamide significantly reduced FID in the low salt diet-fed group, whereas FID in the HS group was mediated by NO only. Cyclooxygenase-2 mRNA (but not protein) expression was decreased in the HS and HS + tempol in vivo groups. Hypoxia-inducible factor-1α and VEGF protein levels were increased in the HS group but decreased in the HS + tempol in vivo group. Assessment by direct fluorescence of middle cerebral arteries under flow revealed significantly reduced vascular NO levels and increased superoxide/reactive oxygen species levels in the HS group. These results suggest that HS intake impairs FID and changes FID mechanisms to entirely NO dependent, in contrast to the low-salt diet-fed group, where FID is NO, prostanoid, and epoxyeicosatrienoic acid dependent. These changes were accompanied by increased lipid peroxidation products in the plasma of HS diet-fed rats, increased vascular superoxide/reactive oxygen species levels, and decreased NO levels, together with increased expression of hypoxia-inducible factor-1α and VEGF. NEW & NOTEWORTHY High-salt (HS) diet changes the mechanisms of flow-induced dilation in rat middle cerebral arteries from a combination of nitric oxide-, prostanoid-, and epoxyeicosatrienoic acid-dependent mechanisms to, albeit reduced, a solely nitric oxide-dependent dilation. In vivo reactive oxygen species scavenging restores flow-induced dilation in HS diet-fed rats and ameliorates HS-induced increases in the transcription factor hypoxia-inducible factor-1α and expression of its downstream target genes.

Acute intermittent hypoxia and rehabilitative training following cervical spinal injury alters neuronal hypoxia- and plasticity-associated protein expression.

One of the most promising approaches to improve recovery after spinal cord injury (SCI) is the augmentation of spontaneously occurring plasticity in uninjured neural pathways. Acute intermittent hypoxia (AIH, brief exposures to reduced O2 levels alternating with normal O2 levels) initiates plasticity in respiratory systems and has been shown to improve recovery in respiratory and non-respiratory spinal systems after SCI in experimental animals and humans. Although the mechanism by which AIH elicits its effects after SCI are not well understood, AIH is known to alter protein expression in spinal neurons in uninjured animals. Here, we examine hypoxia- and plasticity-related protein expression using immunofluorescence in spinal neurons in SCI rats that were treated with AIH combined with motor training, a protocol which has been demonstrated to improve recovery of forelimb function in this lesion model. Specifically, we assessed protein expression in spinal neurons from animals with incomplete cervical SCI which were exposed to AIH treatment + motor training either for 1 or 7 days. AIH treatment consisted of 10 episodes of AIH: (5 min 11% O2: 5 min 21% O2) for 7 days beginning at 4 weeks post-SCI. Both 1 or 7 days of AIH treatment + motor training resulted in significantly increased expression of the transcription factor hypoxia-inducible factor-1α (HIF-1α) relative to normoxia-treated controls, in neurons both proximal (cervical) and remote (lumbar) to the SCI. All other markers examined were significantly elevated in the 7 day AIH + motor training group only, at both cervical and lumbar levels. These markers included vascular endothelial growth factor (VEGF), brain-derived neurotrophic factor (BDNF), and phosphorylated and nonphosphorylated forms of the BDNF receptor tropomyosin-related kinase B (TrkB). In summary, AIH induces plasticity at the cellular level after SCI by altering the expression of major plasticity- and hypoxia-related proteins at spinal regions proximal and remote to the SCI. These changes occur under the same AIH protocol which resulted in recovery of limb function in this animal model. Thus AIH, which induces plasticity in spinal circuitry, could also be an effective therapy to restore motor function after nervous system injury.

The mTOR Pathway Regulates PKM2 to Affect Glycolysis in Esophageal Squamous Cell Carcinoma.

Objectives:Esophageal squamous cell carcinoma is a highly prevalent cancer withpoor survival rate and prognosis. Increasing evidence suggests an important role for metabolic regulation in treating esophageal squamous cell carcinoma, but the underlying mechanism remains unclear. The pyruvate kinase M2 isoform is a key enzyme in the energy production process, and the upregulation of pyruvate kinase M2 isoform also plays a crucial role in gene transcription and tumorigenesis. The mammalian target of rapamycin pathway regulates an array of cellular functions, including protein synthesis, metabolism, and cell proliferation. The pyruvate kinase M2 isoform and mammalian target of rapamycin pathways both affect metabolism in cancers, and evidence also suggests that the mammalian target of rapamycin downstream transcription factor hypoxia-inducible factor-1α regulates pyruvate kinase M2 isoform. We therefore investigated the regulatory mechanism among pyruvate kinase M2 isoform, mammalian target of rapamycin, and aerobic glycolysis in esophageal squamous cell carcinoma, hoping to prove that mammalian target of rapamycin pathway regulates pyruvate kinase M2 isoform to affect glycolysis in esophageal squamous cell carcinoma.Methods:Immunohistochemical staining was used to compare pyruvate kinase M2 isoform and phospho-mammalian target of rapamycin expression in 30 human pathological esophageal squamous cell carcinoma sections and 30 nontumoral esophageal tissues. Short hairpin RNA was used to inhibit pyruvate kinase M2 isoform and activate mammalian target of rapamycin, after which we monitored changes in glucose consumption and lactate production. Finally, we determined the expression of pyruvate kinase M2 isoform and the mammalian target of rapamycin downstream transcription factor hypoxia-inducible factor-1α, as well as glucose consumption and lactate production, following the modification of mammalian target of rapamycin expression.Results:Immunohistochemical staining showed that both phospho-mammalian target of rapamycin and pyruvate kinase M2 isoform expression were higher in esophageal squamous cell carcinoma than in nontumor tissues. Glucose consumption and lactate production measurements demonstrated that altering mammalian target of rapamycin and pyruvate kinase M2 isoform levels caused corresponding changes in glycolysis in esophageal squamous cell carcinoma cells. When mammalian target of rapamycin was activated or inhibited, expression of pyruvate kinase M2 isoform and hypoxia-inducible factor-1α as well as glycolysis were altered, indicating that mammalian target of rapamycin regulates pyruvate kinase M2 isoform via the downstream transcription factor hypoxia-inducible factor-1α, thereby affecting glycolysis in esophageal squamous cell carcinoma.Conclusion:Mammalian target of rapamycin pathway promotes aerobic glycolysis in esophageal squamous cell carcinoma by upregulating pyruvate kinase M2 isoform. Both proteins can serve as molecular targets for novel therapeutic strategies.

Nitric Oxide Modulates Macrophage Responses to Mycobacterium tuberculosis Infection through Activation of HIF-1α and Repression of NF-κB.

IFN-γ is essential for control of Mycobacterium tuberculosis infection in vitro and in vivo. However, the mechanisms by which IFN-γ controls infection remain only partially understood. One of the crucial IFN-γ target genes required for control of M. tuberculosis is inducible NO synthase (iNOS). Although NO produced by iNOS is thought to have direct bactericidal activity against M. tuberculosis, the role of NO as a signaling molecule has been poorly characterized in the context M. tuberculosis infection. In this study, we found that iNOS broadly regulates the macrophage transcriptome during M. tuberculosis infection, activating antimicrobial pathways while also limiting inflammatory cytokine production. The transcription factor hypoxia inducible factor-1α (HIF-1α) was recently shown to be critical for IFN-γ-mediated control of M. tuberculosis infection. We found that HIF-1α function requires NO production, and that HIF-1α and iNOS are linked by a positive feedback loop that amplifies macrophage activation. Furthermore, we found that NO inhibits NF-κB activity to prevent hyperinflammatory responses. Thus, NO activates robust microbicidal programs while also limiting damaging inflammation. IFN-γ signaling must carefully calibrate an effective immune response that does not cause excessive tissue damage, and this study identifies NO as a key player in establishing this balance during M. tuberculosis infection.

PD-L1 Overexpression During Endotoxin Tolerance Impairs the Adaptive Immune Response in Septic Patients via HIF1α.

Sepsis, among other pathologies, is an endotoxin tolerance (ET)-related disease. On admission, we classified 48 patients with sepsis into 3 subgroups according to the ex vivo response to lipopolysaccharide. This response correlates with the Acute Physiology and Chronic Health Evaluation (APACHE) II score and the ET degree. Moreover, the ET-related classification determines the outcome of these patients. Programmed cell death-ligand 1 (PD-L1) expression on septic monocytes is also linked with ET status. In addition to the regulation of cytokine production, one of the hallmarks of ET that significantly affects patients with sepsis is T-cell proliferation impairment or a poor switch to the adaptive response. PD-L1/programmed cell death-1 (PD-1) blocking and knockdown assays on tolerant monocytes from both patients with sepsis and the in vitro model reverted the impaired adaptive response. Mechanistically, the transcription factor hypoxia-inducible factor-1α (HIF1α) has been translocated into the nucleus and drives PD-L1 expression during ET in human monocytes. This fact, together with patient classification according to the ex vivo lipopolysaccharide response, opens an interesting field of study and potential personalized clinical applications, not only for sepsis but also for all ET-associated pathologies.

Hyperglycemia-induced degradation of HIF-1α contributes to impaired response of cardiomyocytes to hypoxia

Introduction and ObjectivesCardiovascular disease is the leading cause of mortality and morbidity associated with diabetes. Although impairment of the cell response to hypoxia due to destabilization of the transcription factor hypoxia-inducible factor-1α (HIF-1α), which regulates the expression of genes that help cells to cope with low oxygen tension, has been implicated in diabetes-associated disease, the molecular mechanisms involved remain elusive. It is known that hyperglycemia leads to the enhanced production of methylglyoxal (MGO). Therefore, the main objective of this study was to establish whether MGO leads to the degradation of HIF-1α in cardiomyocytes subjected to hypoxia. MethodsThe mouse atrial cardiomyocyte cell line, HL-1, was exposed to chemical hypoxia with CoCl2 in the absence or presence of MGO. Cell viability was assessed by MTT assay, and levels of HIF-1α and endogenous ubiquitin conjugates were determined by western blotting. Proteasome activity was analyzed using a specific chymotrypsin-like fluorogenic substrate. ResultsThe results obtained indicate that MGO induces time- and dose-dependent degradation of HIF-1α accumulated under hypoxia. Additionally, we show that accumulation of endogenous ubiquitin conjugates in the presence of MGO is associated with decreased proteasome activity. ConclusionTaken together, the results obtained in this study suggest that MGO compromises the ability of cells to adapt to low oxygen tensions, by stimulating the degradation of HIF-1α, likely contributing to the development of diabetes-associated cardiac dysfunction.

Hyperglycemia-induced degradation of HIF-1α contributes to impaired response of cardiomyocytes to hypoxia

Introduction and ObjectivesCardiovascular disease is the leading cause of mortality and morbidity associated with diabetes. Although impairment of the cell response to hypoxia due to destabilization of the transcription factor hypoxia-inducible factor-1α (HIF-1α), which regulates the expression of genes that help cells to cope with low oxygen tension, has been implicated in diabetes-associated disease, the molecular mechanisms involved remain elusive. It is known that hyperglycemia leads to the enhanced production of methylglyoxal (MGO). Therefore, the main objective of this study was to establish whether MGO leads to the degradation of HIF-1α in cardiomyocytes subjected to hypoxia. MethodsThe mouse atrial cardiomyocyte cell line, HL-1, was exposed to chemical hypoxia with CoCl2 in the absence or presence of MGO. Cell viability was assessed by MTT assay, and levels of HIF-1α and endogenous ubiquitin conjugates were determined by western blotting. Proteasome activity was analyzed using a specific chymotrypsin-like fluorogenic substrate. ResultsThe results obtained indicate that MGO induces time- and dose-dependent degradation of HIF-1α accumulated under hypoxia. Additionally, we show that accumulation of endogenous ubiquitin conjugates in the presence of MGO is associated with decreased proteasome activity. ConclusionTaken together, the results obtained in this study suggest that MGO compromises the ability of cells to adapt to low oxygen tensions, by stimulating the degradation of HIF-1α, likely contributing to the development of diabetes-associated cardiac dysfunction.

Regulation of IL-12p40 by HIF controls Th1/Th17 responses to prevent mucosal inflammation.

Intestinal inflammatory lesions are inherently hypoxic, due to increased metabolic demands created by cellular infiltration and proliferation, and reduced oxygen supply due to vascular damage. Hypoxia stabilizes the transcription factor hypoxia-inducible factor-1α (HIF) leading to a coordinated induction of endogenously protective pathways. We identified IL12B as a HIF-regulated gene and aimed to define how the HIF-IL-12p40 axis influenced intestinal inflammation. Intestinal lamina propria lymphocytes (LPL) were characterized in wild-type and IL-12p40-/- murine colitis treated with vehicle or HIF-stabilizing prolyl-hydroxylase inhibitors (PHDi). IL12B promoter analysis was performed to examine hypoxia-responsive elements. Immunoblot analysis of murine and human LPL supernatants was performed to characterize the HIF/IL-12p40 signaling axis. We observed selective induction of IL-12p40 following PHDi-treatment, concurrent with suppression of Th1 and Th17 responses in murine colitis models. In the absence of IL-12p40, PHDi-treatment was ineffective. Analysis of the IL12B promoter identified canonical HIF-binding sites. HIF stabilization in LPLs resulted in production of IL-12p40 homodimer which was protective against colitis. The selective induction of IL-12p40 by HIF-1α leads to a suppression of mucosal Th1 and Th17 responses. This HIF-IL12p40 axis may represent an endogenously protective mechanism to limit the progression of chronic inflammation, shifting from pro-inflammatory IL-12p70 to an antagonistic IL-12p40 homodimer.

The metabolic bone disease associated with the Hyp mutation is independent of osteoblastic HIF1α expression

Fibroblast growth factor-23 (FGF23) controls key responses to systemic phosphate increases through its phosphaturic actions on the kidney. In addition to stimulation by phosphate, FGF23 positively responds to iron deficiency anemia and hypoxia in rodent models and in humans. The disorder X-linked hypophosphatemia (XLH) is characterized by elevated FGF23 in concert with an intrinsic bone mineralization defect. Indeed, the Hyp mouse XLH model has disturbed osteoblast to osteocyte differentiation with altered expression of a wide variety of genes, including FGF23. The transcription factor Hypoxia inducible factor-1α (HIF1α) has been implicated in regulating FGF23 production and plays a key role in proper bone cell differentiation. Thus the goals of this study were to determine whether HIF1α activation could influence FGF23, and to test osteoblastic HIF1α production on the Hyp endocrine and skeletal phenotypes in vivo. Treatment of primary cultures of osteoblasts/osteocytes and UMR-106 cells with the HIF activator AG490 resulted in rapid HIF1α stabilization and increased Fgf23 mRNA (50–100 fold; p<0.01–0.001) in a time- and dose-dependent manner. Next, the Phex gene deletion in the Hyp mouse was bred onto mice with a HIF1α/Osteocalcin (OCN)-Cre background. Although HIF1α effects on bone could be detected, FGF23-related phenotypes due to the Hyp mutation were independent of HIF1α in vivo. In summary, FGF23 can be driven by ectopic HIF1α activation under normal iron conditions in vitro, but factors independent of HIF1α activity after mature osteoblast formation are responsible for the disease phenotypes in Hyp mice in vivo.

Trypanosoma brucei metabolite indolepyruvate decreases HIF-1α and glycolysis in macrophages as a mechanism of innate immune evasion

The parasite Trypanasoma brucei causes African trypanosomiasis, known as sleeping sickness in humans and nagana in domestic animals. These diseases are a major burden in the 36 sub-Saharan African countries where the tsetse fly vector is endemic. Untreated trypanosomiasis is fatal and the current treatments are stage-dependent and can be problematic during the meningoencephalitic stage, where no new therapies have been developed in recent years and the current drugs have a low therapeutic index. There is a need for more effective treatments and a better understanding of how these parasites evade the host immune response will help in this regard. The bloodstream form of T. brucei excretes significant amounts of aromatic ketoacids, including indolepyruvate, a transamination product of tryptophan. This study demonstrates that this process is essential in bloodstream forms, is mediated by a specialized isoform of cytoplasmic aminotransferase and, importantly, reveals an immunomodulatory role for indolepyruvate. Indolepyruvate prevents the LPS-induced glycolytic shift in macrophages. This effect is the result of an increase in the hydroxylation and degradation of the transcription factor hypoxia-inducible factor-1α (HIF-1α). The reduction in HIF-1α levels by indolepyruvate, following LPS or trypanosome activation, results in a decrease in production of the proinflammatory cytokine IL-1β. These data demonstrate an important role for indolepyruvate in immune evasion by T. brucei.

Loss of smooth muscle cell hypoxia inducible factor-1α underlies increased vascular contractility in pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is an often fatal disease with limited treatment options. Whereas current data support the notion that, in pulmonary artery endothelial cells (PAECs), expression of transcription factor hypoxia inducible factor-1α (HIF-1α) is increased, the role of HIF-1α in pulmonary artery smooth muscle cells (PASMCs) remains controversial. This study investigates the hypothesis that, in PASMCs from patients with PAH, decreases in HIF-1α expression and activity underlie augmented pulmonary vascular contractility. PASMCs and tissues were isolated from nonhypertensive control patients and patients with PAH. Compared with controls, HIF-1α and Kv1.5 protein expression were decreased in PAH smooth muscle cells (primary culture). Myosin light chain (MLC) phosphorylation and MLC kinase (MLCK) activity-major determinants of vascular tone-were increased in patients with PAH. Cofactors involved in prolyl hydroxylase domain activity were increased in PAH smooth muscle cells. Functionally, PASMC contractility was inversely correlated with HIF-1α activity. In PASMCs derived from patients with PAH, HIF-1α expression is decreased, and MLCK activity, MLC phosphorylation, and cell contraction are increased. We conclude that compromised PASMC HIF-1α expression may contribute to the increased tone that characterizes pulmonary hypertension.-Barnes, E. A., Chen, C.-H., Sedan, O., Cornfield, D. N. Loss of smooth muscle cell hypoxia inducible factor-1α underlies increased vascular contractility in pulmonary hypertension.

Anti-Warburg effect of rosmarinic acid via miR-155 in colorectal carcinoma cells.

The Warburg effect, glycolytic production of ATP under aerobic conditions, is found to be a universal feature of most cancer cells. Our study was aimed to determine whether rosmarinic acid (RA) had the anti-Warburg effect activity against colorectal carcinoma. Furthermore, the mechanism for the anti-Warburg effect by RA would be investigated. In our study, we found that RA suppressed glucose consumption and lactate generation in colorectal carcinoma cells; meanwhile, RA inhibited the expression of transcription factor hypoxia-inducible factor-1α (HIF-1α) that affects the glycolytic pathway. Chronic inflammation is a key promoting factor of the Warburg effect. As we supposed, the present study also showed that RA could not only repress proinflammatory cytokines using enzyme-linked immunosorbent assay but it could also suppress microRNAs related to inflammation by real-time PCR. Therefore, we proposed that RA may inhibit the Warburg effect by suppressing the inflammatory response of colorectal carcinoma cells. Recent studies have provided evidence that miR-155 was an important mediator between inflammation and carcinogenesis. We further showed that miR-155 acted to repress the Warburg effect through the mechanism of inactivating the IL-6/STAT3 pathway. Above all, RA might be a potential therapeutic agent against colorectal carcinoma.

Klebsiella pneumoniae Siderophores Induce Inflammation, Bacterial Dissemination, and HIF-1α Stabilization during Pneumonia.

ABSTRACTKlebsiella pneumoniae is a Gram-negative pathogen responsible for a wide range of infections, including pneumonia and bacteremia, and is rapidly acquiring antibiotic resistance. K. pneumoniae requires secretion of siderophores, low-molecular-weight, high-affinity iron chelators, for bacterial replication and full virulence. The specific combination of siderophores secreted by K. pneumoniae during infection can impact tissue localization, systemic dissemination, and host survival. However, the effect of these potent iron chelators on the host during infection is unknown. In vitro, siderophores deplete epithelial cell iron, induce cytokine secretion, and activate the master transcription factor hypoxia inducible factor-1α (HIF-1α) protein that controls vascular permeability and inflammatory gene expression. Therefore, we hypothesized that siderophore secretion by K. pneumoniae directly contributes to inflammation and bacterial dissemination during pneumonia. To examine the effects of siderophore secretion independently of bacterial growth, we performed infections with tonB mutants that persist in vivo but are deficient in siderophore import. Using a murine model of pneumonia, we found that siderophore secretion by K. pneumoniae induces the secretion of interleukin-6 (IL-6), CXCL1, and CXCL2, as well as bacterial dissemination to the spleen, compared to siderophore-negative mutants at an equivalent bacterial number. Furthermore, we determined that siderophore-secreting K. pneumoniae stabilized HIF-1α in vivo and that bacterial dissemination to the spleen required alveolar epithelial HIF-1α. Our results indicate that siderophores act directly on the host to induce inflammatory cytokines and bacterial dissemination and that HIF-1α is a susceptibility factor for bacterial invasion during pneumonia.

HIF-1α Is an Essential Mediator of IFN-γ-Dependent Immunity to Mycobacterium tuberculosis.

The cytokine IFN-γ coordinates macrophage activation and is essential for control of pathogens, including Mycobacterium tuberculosis However, the mechanisms by which IFN-γ controls M. tuberculosis infection are only partially understood. In this study, we show that the transcription factor hypoxia-inducible factor-1α (HIF-1α) is an essential mediator of IFN-γ-dependent control of M. tuberculosis infection both in vitro and in vivo. M. tuberculosis infection of IFN-γ-activated macrophages results in a synergistic increase in HIF-1α protein levels. This increase in HIF-1α levels is functionally important, as macrophages lacking HIF-1α are defective for IFN-γ-dependent control of infection. RNA-sequencing demonstrates that HIF-1α regulates nearly one-half of all IFN-γ-inducible genes during infection of macrophages. In particular, HIF-1α regulates production of important immune effectors, including inflammatory cytokines and chemokines, eicosanoids, and NO. In addition, we find that during infection HIF-1α coordinates a metabolic shift to aerobic glycolysis in IFN-γ-activated macrophages. We find that this enhanced glycolytic flux is crucial for IFN-γ-dependent control of infection in macrophages. Furthermore, we identify a positive feedback loop between HIF-1α and aerobic glycolysis that amplifies macrophage activation. Finally, we demonstrate that HIF-1α is crucial for control of infection in vivo as mice lacking HIF-1α in the myeloid lineage are strikingly susceptible to infection and exhibit defective production of inflammatory cytokines and microbicidal effectors. In conclusion, we have identified HIF-1α as a novel regulator of IFN-γ-dependent immunity that coordinates an immunometabolic program essential for control of M. tuberculosis infection in vitro and in vivo.

Inhibition of HIF-1α enhances anti-tumor effects of dendritic cell-based vaccination in a mouse model of breast cancer.

Considerable evidence shows that the tumor microenvironment is an active participant in preventing immunosurveillance and limiting the efficacy of anticancer therapies. Hypoxia is a prominent characteristic of the solid tumor microenvironment. The transcription factor hypoxia-inducible factor-1α (HIF-1α) is an important mediator of hypoxic response of tumor cells that modulates the expression of specific genes involved in tumor immunosuppression. Using a 4T1 breast cancer model, we show that in vivo administration of PX-478, an inhibitor of oxygen-sensitive HIF-1α, led to reduced expression of Foxp3 and VEGF transcript and/or protein, molecules that are directly controlled by HIF-1. When combined with dendritic cell (DC)-based vaccination, HIF-1α inhibition resulted in an augmented cytotoxic T lymphocyte effector function, improved proliferation status of T cells, increased production of inflammatory cytokine IFN-γ, as well as reduced regulatory function of T cells in association with slower tumor growth. Taken together, our findings indicate that the use of HIF-1α inhibition provides an immune adjuvant activity, thereby improves the efficacy of tumor antigen-based DC vaccine.

STAT3 and mTOR: co-operating to drive HIF and angiogenesis.

Our understanding of angiogenic signalling has been significantly enhanced through studies of a rare genetic disorder called Tuberous Sclerosis Complex (TSC). TSC patients are predisposed to highly vascularised tumours, where renal angiomyolipomas produce high levels of vascular endothelial growth factor (VEGF) that can be readily detected. It is well established that VEGF is driven through hypoxic signalling, with the transcription factor hypoxia inducible factor-1α (HIF-1α) playing a crucial role in its expression. Early studies using cell line models of TSC uncovered that the mammalian target of rapamycin complex 1 (mTORC1) is a key mediator of HIF-1α synthesis, and highlighted the anti-angiogenic properties of mTORC1 inhibitors [1]. Herein we review our recent findings characterising mTORC1 mediated regulation of HIF-1α and discuss the clinical implications of our work. We demonstrated that mTORC1 drives HIF-1α expression via three mechanisms, promoting not only the transcription of HIF-1α mRNA via signal transducer and activator of transcription 3 (STAT3), but also its translation via both eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and ribosomal protein S6 kinase 1 (S6K1). This drive in HIF-1α activity downstream of mTORC1 explains why the tumours which present in TSC are so heavily vascularised, and accounts for the anti-tumorigenic properties of mTOR inhibitors used in this setting. In concordance with this, we observe a 10 fold-increase in HIF-1α transcriptional activity under hypoxia with TSC2 loss, highlighting the significant impact mTORC1 activation can have on HIF-1α. Whilst mTORC1 can promote the transcriptional activity of STAT3 through direct phosphorylation of Ser727, STAT3 is also subject to regulation from a number of different cytokines and growth factors which signal through the receptor tyrosine kinase JAK2 [2]. Both JAK2/STAT3 and mTORC1 signalling pathways are frequently activated in a wide range of malignancies and converge at the level of HIF-1α (see Figure ​Figure1).1). Whilst mTOR inhibitors are effective at blocking Ser727 phosphorylation of STAT3, we were able to completely abolish HIF-1α expression by targeting both the JAK2-mediated Tyr705 phosphorylation site and the mTORC1-mediated Ser727 site. Our work indicates that targeting STAT3 in parallel to mTORC1 could enhance the anti-angiogenic and anti-tumorigenic properties of mTOR inhibitors that are currently in clinical use [3]. Figure 1 Multifaceted regulation of HIF-1α/VEGF-A via mTORC1 and STAT3 Growth of tumours in renal cell carcinoma (RCC) is highly dependent on mTORC1, HIF and VEGF which drive a pro-angiogenic response. In the microenvironment of the kidney, angiogenic signalling is crucial for metabolic transformation and malignancy. Although there has been much investment into drug discovery and the development of inhibitors that directly inhibit HIF, none of these compounds are currently suitable for clinical use. Consequently, current angiogenic therapies have been mainly restricted to inhibition of mTORC1 and the VEGF-receptor (VEGFR). Given that our work now positions STAT3 as an immediate upstream regulator of HIF, drug targeting of STAT3 may be an alternative therapeutic strategy for the treatment of vascularized tumours. We also identified STAT3 as a common point of convergence for many receptor tyrosine kinases involved in the angiogenic response within Neurofibromatosis 1 (NF1) associated malignant peripheral nerve sheath tumours (MPNSTs) [4], reflecting the wider implications of this work. We uncovered that STAT3 inhibition or shRNA knockdown was sufficient to completely ablate expression of HIF-1α, HIF-2α and VEGF-A. Previous studies indicated that rapamycin repressed the growth of NF1-associated malignancies, but with minimal effects upon HIF-1α expression indicating that STAT3 primarily drives HIF-1α activity in NF1 [5]. STAT3 is considered an oncogene and mediates a range of cellular processes associated with tumourigenesis, increasing its appeal as a potential therapeutic target [2]. In keeping with this, knockdown of STAT3 inhibited cell migration and blocked tumour spheroid formation in a range of different MPNST cell lines tested [4]. A current limitation of anti-angiogenic therapies targeting the VEGF receptor (VEGFR) is acquired drug resistance. This occurs through activation of compensatory signalling pathways coordinating tumour revascularisation, where a multitude of pro-angiogenic factors (such as VEGF, FGFs, EGF, and IL-8 (as well as others)) signal through a network of target receptor tyrosine kinases (RTK) to elicit the pro-angiogenic response. Consequently, angiogenic signalling is highly flexible and driven through cross-talk between multiple RTKs. This level of flexibility hampers current tyrosine kinase inhibitor therapies [6]. Given that STAT3 is a common downstream target of multiple RTKs, acquired resistance may be less of a problem in this setting. STAT3 is also fairly indispensable in normal cellular function, suggesting that the side effect profile of STAT3 inhibitors should be low. Collectively, our work highlights the need for the development of clinically available STAT3 inhibitors for cancer therapy.

HIF-1α and growth plate development: what we really know.

Adaptation to low oxygen tension or hypoxia is a critical event in development and tissue homeostasis. Studies by us and others have shown that the fetal growth plate is an avascular tissue with a gradient of oxygenation, and the transcription factor hypoxia-inducible factor-1α (HIF-1α) is essential for its development. In this brief review, we will summarize our current understanding of the role of HIF-1α in fetal growth plate development, and we will discuss yet unanswered questions in the field of hypoxia and endochondral bone formation.

Transcription regulates HIF-1α expression in CD4(+) T cells.

The transcription factor hypoxia inducible factor-1α (HIF-1α) mediates the metabolic adaptation of cells to hypoxia and T-helper cell fate. However, HIF-1α regulation in CD4(+) T cells (T cells) remains elusive. Here we observed that depletion of oxygen (O2⩽2%) alone was not sufficient to induce HIF-1α expression in T cells. However, when hypoxic T cells were stimulated, HIF-1α was expressed and this was dependent on nuclear factor-κB- and nuclear factor of activated T cell (NFAT)-mediated transcriptional upregulation of Hif-1α mRNA. HIF-1α upregulation could be blocked by drugs inhibiting NF-κB, NFAT or mammalian target of rapamycin precluding CD4(+) T-cell stimulation or translation in T cells, as well as by blocking transcription. CD3, CD28, phorbol-12-myristat-13-acetat (PMA) or ionomycin-stimulated T cells did not express HIF-1α under normoxic conditions. In conclusion, regulation of HIF-1α expression in CD4(+) T cells in hypoxia gravely relies on its transcriptional upregulation and subsequent enhanced protein stabilization.