Role Of Hypoxia-inducible Transcription Factors Research Articles

Bioenergetic alteration in gastrointestinal cancers: The good, the bad and the ugly.

Cancer cells exhibit metabolic reprogramming and bioenergetic alteration, utilizing glucose fermentation for energy production, known as the Warburg effect. However, there are a lack of comprehensive reviews summarizing the metabolic reprogramming, bioenergetic alteration, and their oncogenetic links in gastrointestinal (GI) cancers. Furthermore, the efficacy and treatment potential of emerging anticancer drugs targeting these alterations in GI cancers require further evaluation. This review highlights the interplay between aerobic glycolysis, the tricarboxylic acid (TCA) cycle, and oxidative phosphorylation (OXPHOS) in cancer cells, as well as hypotheses on the molecular mechanisms that trigger this alteration. The role of hypoxia-inducible transcription factors, tumor suppressors, and the oncogenetic link between hypoxia-related enzymes, bioenergetic changes, and GI cancer are also discussed. This review emphasizes the potential of targeting bioenergetic regulators for anti-cancer therapy, particularly for GI cancers. Emphasizing the potential of targeting bioenergetic regulators for GI cancer therapy, the review categorizes these regulators into aerobic glycolysis/ lactate biosynthesis/transportation and TCA cycle/coupled OXPHOS. We also detail various anti-cancer drugs and strategies that have produced pre-clinical and/or clinical evidence in treating GI cancers, as well as the challenges posed by these drugs. Here we highlight that understanding dysregulated cancer cell bioenergetics is critical for effective treatments, although the diverse metabolic patterns present challenges for targeted therapies. Further research is needed to comprehend the specific mechanisms of inhibiting bioenergetic enzymes, address side effects, and leverage high-throughput multi-omics and spatial omics to gain insights into cancer cell heterogeneity for targeted bioenergetic therapies.

Hypoxia-inducible transcription factor-1\u03b1 inhibition by topotecan protects against lipopolysaccharide-induced inflammation and apoptosis of cardiomyocytes

BackgroundMyocarditis, an inflammatory disease of the myocardium, is a serious hazard to human life due to the expansion of inflammatory lesions in the myocardium. The aim of this study was to investigate the role of hypoxia-inducible transcription factor (HIF)-1α and its inhibitor topotecan in the pathogenesis of myocarditis.MethodsH9c2 cardiomyoblasts was stimulated with lipopolysaccharide (LPS) to simulate myocarditis model in vitro. The levels of myocardial damage markers were determined using commercially available kits. Western blotting was used to evaluate HIF-1α expression after LPS challenge. Then, after HIF-1α silencing, the contents of inflammatory factors were determined with enzyme-linked immunosorbent assay (ELISA). Cell viability was tested by means of a cell counting kit-8 (CCK-8) assay. Cell apoptosis was assessed by flow cytometry, and the expression of apoptotic proteins was examined using western blot analysis. Subsequently, HIF-1α was overexpressed and topotecan was employed to treat H9c2 cells under LPS exposure condition. The biological functions were detected again.ResultsLPS significantly elevated the levels of lactate dehydrogenase (LDH), creatine kinase-MB (CK-MB) and cardiac troponin-I (cTn-I) in supernatant of H9c2 cell lysates. Additionally, LPS led to the notably upregulated expression of HIF-1α. HIF-1α-knockdown markedly decreased the concentrations of tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-8 compared with the LPS-induced group. Moreover, the cell viability was conspicuously enhanced and cell apoptotic ratio was remarkably reduced, accompanied by downregulated expression of Bax, Bim, caspase 3 and caspase 9 after HIF-1α silencing. Consistently, HIF-1α gain-of-function significantly promoted the production of inflammatory cytokines and cell apoptosis, which was partially counteracted by topotecan administration.ConclusionTo conclude, these findings demonstrated that HIF-1α inhibition by topotecan ameliorates LPS-induced myocarditis in vitro, providing a new approach in the treatment of myocarditis.

Preconditioned suppression of prolyl-hydroxylases attenuates renal injury but increases mortality in septic murine models.

Septic conditions contribute to tissue hypoxia, potentially leading to multiple organ failure, including acute kidney injury. The regulation of cellular adaptation to low oxygen levels is regulated by hypoxia-inducible transcription factors (HIFs). While the role of HIFs in ischaemia/reperfusion is more studied, their function in sepsis-induced renal injury is not well characterized. In this study, we investigated whether pharmacological activation of HIFs by suppression of prolyl-hydroxylases (PHDs) protects against septic acute kidney injury. Two models of sepsis-caecal ligation and punction and peritoneal contamination and infection-were induced on 12-week-old C57BL6/J mice. Pharmacological inhibition of PHDs, leading to HIF activation, was achieved by intraperitoneal application of 3,4-dihydroxybenzoate (3,4-DHB) before sepsis. A quantitative real-time reverse transcription polymerase chain reaction, immunohistology and enzyme-linked immunosorbent assays were utilized to detect gene expression, renal protein levels and renal functional parameters, respectively. Tissue morphology was analysed by periodic acid-Schiff reaction. Early kidney injury was estimated by kidney injury molecule-1 analyses. Apoptosis was detected in situ by terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling stain. The systemic effect of 3,4-DHB pretreatment in sepsis was analysed by 72-h survival studies. Pharmacological activation of HIFs before sepsis induction attenuated sepsis-related vacuolization and dilation of the proximal tubules, reduced tubular apoptosis and correlated to lower T-cell infiltration in renal tissue compared with the non-treated septic animals. PHD suppression elevated the basal renal HIF-1α expression and basal plasma concentrations of HIF targets erythropoietin and vascular endothelial growth factor. Whereas it preserved renal structure in both models, it improved renal function in a model-dependent manner. Moreover, inhibition of PHDs led to increased mortality in both models. Analysis of liver function showed increased organ destruction with massive glycogen loss and hepatocyte's apoptosis due to 3,4-DHB administration before sepsis induction. In summary, the pharmacological activation of HIFs by 3,4-DHB administration, although it showed renoprotective effects in sepsis-related kidney injury, induced more severe problems in other organs such as the liver during sepsis, leading to increased mortality.

Acute hypoxia modifies regulation of neuroglobin in the neonatal mouse brain

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

Hypoxia and Inflammation

To the Editor: In their review article on hypoxia and inflammation, Eltzschig and Carmeliet (Feb. 17 issue)1 were thorough in promoting the role of hypoxia-inducible transcription factor (HIF) and related mechanisms to regain homeostasis in hypoxic tissue environments. Although very exacting in its discussion, the review unfortunately failed to include other, equally important mechanisms that lead to inflammation from hypoxic–ischemic reperfusion injury. I am referring in part to the importance of the inflammasome, and in particular to the NLRP3 inflammasome that responds to danger-associated molecular patterns (e.g., urates, free ATP) resulting from nucleoprotein catabolism induced by such injury. Such stimulation leads to secretion of proinflammatory cytokines, in particular interleukin-1β,2 resulting in neutrophilic inflammation. The NLRP3 inflammasome mechanism was first discovered in autosomal autoinflammatory syndromes, namely cryopyrin-associated periodic syndromes, and these disorders have responded beautifully to interleukin-1–targeted therapies. Now there is current interest among several pharmaceutical companies to test similar therapies in inflammatory disorders caused by hypoxic–ischemic reperfusion injury.2-4

Enhanced leukocyte HIF-1α and HIF-1 DNA binding in humans after rapid ascent to 4300 m

Hypoxia plays a crucial role in the pathogenesis of a multitude of diseases and clinical conditions such as cancer, diabetes, cardiovascular disease, stroke, pulmonary disease, inflammation, organ transplant, and wound healing. Investigations into the role of hypoxia-inducible transcription factor (HIF) in disease development have been conducted with the basic premise that HIF is activated in vivo during hypoxia in humans, yet this basic physiologic premise has never verified. Thus, we hypothesized that HIF-1 DNA binding would be enhanced in vivo in humans in response to acute global hypoxia. Fourteen human subjects were exposed to normoxia (1600 m) and hypoxia (4300 m, ≈ 12% O 2) in a hypobaric hypoxic chamber (8 h). HIF-1 DNA binding and HIF-1α protein were evaluated in circulating leukocytes. Oxidative markers were evaluated by plasma metabolomics using nuclear magnetic resonance and by urinary 15-F 2t-isoprostane concentrations. Leukocyte HIF-1 DNA binding was increased ( p = 0.007) and HIF-1α was greater during hypoxia compared to normoxia. Circulating total glutathione was reduced by 35% ( p = 0.001), and lactate and succinate were increased by 29 and 158%, respectively ( p = 0.007 and 0.001), as were urinary 15-F 2t-isoprostanes ( p = 0.037). HIF-1 DNA binding and HIF-1α were elevated in vivo in leukocytes of healthy human subjects exposed to 12% oxygen, in association with plasma and urinary markers of hypoxic stress.

Hypoxia-induced secretion of macrophage migration-inhibitory factor from MCF-7 breast cancer cells is regulated in a hypoxia-inducible factor-independent manner

The cytokine MIF is over-expressed in tumors and is associated with tumor proliferation, angiogenesis and metastasis. Hypoxia, a hallmark feature of tumors, increases MIF expression from tumor cells. We examined the role of hypoxia-inducible transcription factors on MIF secretion from MCF-7 breast carcinoma cells. Secretion of MIF was induced by hypoxia after 24 h but up-regulation of MIF mRNA was minimal. Inhibition of HIF-1α, HIF-2α, NF-κB and C/EBPβ using siRNA had no effect on hypoxia-induced MIF secretion. However, inhibition of transcription and translation significantly decreased MIF production, suggesting that hypoxia-induced secretion of MIF in MCF-7 cells is via an alternative pathway.

Role of Hypoxia-Inducible Transcription Factors 1α and 2α in the Regulation of Plasminogen Activator Inhibitor-1 Expression in a Human Trophoblast Cell Line

The plasminogen activator inhibitors (PAIs) play critical roles in regulating hemostatic and invasive functions of trophoblasts through suppression of plasmin-dependent fibrinolysis and extracellular matrix degradation. The expression of PAI-1 is increased under hypoxic conditions, although the mechanism remains incompletely understood. In the current study we used HTR-8/SVneo cells, a first trimester extravillous trophoblast cell line, and siRNA technology to examine the role of hypoxia-inducible transcription factors (HIFs)-1α and -2α in the regulation of PAI-1 expression. Using serum-containing and serum-free media culture media it was initially noted that levels of PAI-1, but not PAI-2 protein, were markedly induced by hypoxic (2–3% oxygen) treatment. Under hypoxic conditions, Western blotting revealed that the presence of siRNAs to HIF-1α and HIF-2α suppressed expression of their respective proteins, whereas treatment with non-targeting and cyclophilin B siRNAs did not. Importantly, incubation with siRNA to HIF-1α or HIF-2α alone reduced PAI-1 protein levels to a similar extent, with the combined treatment inducing a more profound effect. The presence of HIF siRNAs reduced levels of PAI-1 mRNA as measured by quantitative real-time PCR, indicating that HIF-1α and HIF-2 α regulate PAI-1 expression at a transcriptional level. These results indicate that both HIF-1α and HIF-2α play important and similar roles in hypoxia-mediated stimulation of PAI-1 expression in HTR-8/SVneo cells. Our findings provide insight into the physiological regulation of trophoblast PAI-1 expression in early pregnancy when placental oxygen levels are low, as well as a mechanism for over-expression of placental PAI-1 noted in pregnancies with preeclampsia.

Role of hypoxia-inducible transcription factors (HIFs) in oxygen-dependent modulation of IGF binding protein–1 biosynthesis in primary rat hepatocytes

Higher levels of IGFBP–1 mRNA are expressed in the less aerobic perivenous zone of the liver. Since gradients in oxygen tension (pO2) may contribute to zonated gene expression, the influence of arterial and venous pO2 on IGFBP–1 biosynthesis was studied in primary cultures of rat hepatocytes. Maximal IGFBP–1 mRNA and protein levels were observed under venous pO2 whereas less than 30% of maximal levels were observed under arterial pO2. In contrast, the expression of IGFBP–4 was greatest under arterial pO2, indicating that this effect of hypoxia on IGFBP–1 gene expression is specific. The response to hypoxia appeares to involve reactive oxygen species, since treatment with H2O2 results in a dose-dependent decrease of IGFBP–1 mRNA levels under venous pO2, whereas IGFBP–1 mRNA expression under arterial pO2 was not affected. Inhibition of the hypoxia-dependent IGFBP–1 mRNA induction by actinomycin D indicates that this effect is mediated at the level of gene transcription, inhibition of IGFBP–1 mRNA by the iron chelator desferrioxamine under both venous and arterial pO2 suggested an involvement of hypoxia-inducible transcription factors (HIF). Transfection experiments demonstrated that especially HIF–3α and HIF–2α, and to a lesser extent HIF–1α, contribute to the induction of IGFBP–1 mRNA expression in isolated hepatocytes, while experiments with vectors for the HIF prolyl hydroxylases (PHD) indicated a major role of PHD–2 in destabilization of HIFs, attenuating the induction of IGFBP–1 under venous pO2. Reporter gene studies indicate that hypoxia stimulates IGFBP–1 expression through a putative HIF response element located ~250 bp upstream from the transcription initiation site. Together, these results support the concept that iron, radical oxygen species, the HIF–2 and –3 as well as the PHD pathways play important roles in mediating effects of hypoxia on IGFBP–1 gene expression in the liver.

Hypoxia-inducible factors: where, when and why?

Hypoxia-inducible factor (HIF) is a family of transcription factors that regulate the homeostatic response to oxygen deprivation during development, physiological adaptation, and pathological processes such as ischemia and neoplasia. Our understanding of the function of different HIF isoforms is being advanced by understanding the processes that regulate their activity, learning where and when they are expressed and what genes they regulate.

Impact of HIF‐1α and HIF‐2α on proliferation and migration of human pulmonary artery fibroblasts in hypoxia

Proliferation of adventitial fibroblasts of small intrapulmonary arteries (FBPA) has been disclosed as an early event in the development of pulmonary hypertension and cor pulmonale in response to hypoxia. We investigated the role of hypoxia-inducible transcription factors (HIF) in human FBPA exposed to hypoxia. Primary cultures of FBPA displayed a strong mitogenic response to 24 h hypoxia, whereas the rate of apoptosis was significantly suppressed. In addition, the migration of FBPA was strongly increased under hypoxic conditions but not the expression of alpha-smooth muscle actin. Hypoxia induced a marked up-regulation (protein level) of both HIF-1alpha and HIF-2alpha, alongside with nuclear translocation of these transcription factors. Specific inhibition of either HIF-1alpha or HIF-2alpha was achieved by RNA interference technology, as proven by HIF-1alpha and HIF-2alpha mRNA and protein analysis and expression analysis of HIF downstream target genes. With the use of this approach, the hypoxia-induced proliferative response of the FBPA was found to be solely HIF-2alpha dependent, whereas the migratory response was significantly reduced by both HIF-1alpha and HIF-2alpha interference. In conclusion, HIF up-regulation is essential for hypoxic cellular responses in human pulmonary artery adventitial fibroblasts such as proliferation and migration, mimicking the pulmonary hypertensive phenotype in vivo. Differential HIF subtype dependency was noted, with HIF-2alpha playing a predominant role, which may offer future intervention strategies.

Hypoxic pulmonary artery fibroblasts trigger proliferation of vascular smooth muscle cells: role of hypoxia-inducible transcription factors.

Chronic lung hypoxia causes vascular remodeling with pulmonary artery smooth muscle cell (SMCPA) hyperplasia, resulting in pulmonary hypertension and cor pulmonale. We investigated SMCPA and pulmonary artery adventitial fibroblasts (FBPA) for their proliferative response to hypoxia. Strong SMCPA growth occurred under hypoxic conditions in SMCPA/FBPA co-cultures, but not in SMCPA monocultures. SMCPA growth was fully reproduced by transferring serum-free supernatant from hypoxic cultured FBPA to normoxic SMCPA. Hypoxia-inducible-transcription-factor subtypes (HIF-1alpha, HIF-2alpha, HIF-3alpha) and its dependent target genes, carrying the hypoxia-responsive-element as regulatory component, were strongly activated in both hypoxic FBPA and SMCPA. HIF-transcription-factor decoy technique, employed to FBPA during hypoxic culturing, blocked the mitogenic activity of FBPA conditioned medium on SMCPA. The data suggest that hypoxia-driven gene regulation in pulmonary artery fibroblasts results in a mitogenic stimulus on adjacent pulmonary artery smooth muscle cells, and HIF-transcription-decoy may offer a new therapeutic approach to suppress these events.