Passing the baton: the HIF switch
ABSTRACT Hypoxia is an inadequate oxygen supply to tissues and cells, which can restrict their function. The hypoxic response is primarily mediated by the hypoxia-inducible transcription factors, HIF-1 and HIF-2, which have both overlapping and unique target genes. HIF target gene activation is highly context specific and is not a reliable indicator of which HIF-α isoform is active. For example, in some cell lines, the individual HIFs have specific temporal and functional roles: HIF-1 drives the initial response to hypoxia (<24h) and HIF-2 drives the chronic response (>24h). Here, we review the significance of the HIF switch and the relation between HIF-1 and HIF-2 under both physiological and pathophysiological conditions.
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ABSTRACT: GPx8 is a mammalian Cys-glutathione peroxidase of the endoplasmic reticulum membrane, involved in protein folding. Its regulation is mostly unknown. We addressed both, functionality of two hypoxia response elements (HREs) within the promoter, GPx8-HRE1 and GPx8-HRE2 and the GPx8 physiological role. In HeLa cells, treatment with HIFα stabilizers, such as diethyl succinate (DES) or 2-2′–bipyridyl (BP) induces GPx8 mRNA 1.5 fold. Luciferase activity of pGL3GPx8wt, containing a fragment of the GPx8 promoter including the two HREs, is also induced by DES/BP or by overexpressing either individual HIFα subunit. Mutating GPx8-HRE1 within pGL3GPx8wt resulted in a significantly higher inhibition of luciferase activity than mutating GPx8-HRE2. EMSA analysis showed that both HREs exhibit enhanced binding to a nuclear extract from DES/BP-treated cells, with stronger binding by GPx8-HRE1. In DES-treated cells transfected with pGL3GPx8wt or mutants thereof, silencing of HIF2α, but not HIF1α, abolishes luciferase activity. Thus GPx8 is a novel HIF target preferentially responding to HIF2α binding at its two novel functional GPx8-HREs, with GPx8-HRE1 playing the major role. FGF treatment increases GPx8 mRNA expression and reporter gene experiments indicate that induction occurs via HIF. Comparing the effect of depleting GPx8 on the downstream effectors of FGF or insulin signaling, revealed that absence of GPx8 results in a 16 or 12 fold increase of phosphorylated ERK 1/2 - by FGF or insulin treatment respectively. Furthermore, in GPx8 depleted cells, phosphorylation of AKT by insulin treatment increases 2.5 fold. We suggest that induction of GPx8 expression by HIF slows down proliferative signaling during hypoxia and/or growth stimulation through receptor tyrosine kinases.Free Radical Biology and Medicine 01/2015; DOI:10.1016/j.freeradbiomed.2014.12.020 · 5.71 Impact Factor
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ABSTRACT: CONTENTS 1. Introduction 1 1.1. Brain tumor glioblastoma multiforme (GBM) 1 1.2. Cancer stem cell (CSC) paradigm 2 1.3. GBM stem-like cells (GSCs) 4 2. Isolation methodologies of GSCs 4 2.1. SP and ABC transporters 5 2.2. Isolation based on biomarkers 5 2.2.1. CD133 5 2.2.2. ALDH 5 2.2.3. Aptamers 5 3. Enrichment of GSC cultures 6 3.1. Effects of medium and substrates 6 3.2. Effects of hypoxia 7 3.3. Effects of endothelial cells 8 3.4. Effects of pericyte cells 9 3.5. Effects of immunosuppressed animals 9 4. Validation of the GSC phenotype 10 4.1. Sphere-formation assay 10 4.2. Tumor formation in vivo 10 4.3. Expression of biomarkers 11 4.4. Chemoresistance assay 11 4.5. Differentiation assay 12 5. Conclusions 12 Acknowledgments 13 References 13 Abstract: Glioblastoma (GBM) stem-like cells (GSCs) represent the most undifferentiated state of malignant cells with distinct biological characteristics. The fraction of these cells within a glial brain tumor, ranging from 2–30%, is correlating with the increasing WHO stage and poor prognosis of patients' survival. GSCs represent the least vulnerable, thus most preferential target cell population to be exposed to various therapeutic modalities, although the underlying mechanisms of this resistance are not yet fully understood. For the development of GSC-targeting therapies, further in depth studies are needed using enriched and stable GSC cell populations. Here, we discuss the current approaches of GSC isolation and validation based on expression of stemness and oncogenic markers as well as on functional assays. The enrichment of GSC phenotypes in established cell lines and/or primary tumor cultures, achieved by different strategies, is reviewed, providing a comprehensive comparison of selected studies and contemplating the characterization of the plethora of variants of reported GBM population exhibiting the GSC phenotype. ABBREVIATIONS: Aldehyde dehydrogenase (ALDH) Brain tumor initiating cells (BTIC) Cancer stem cell (CSC) Epidermal growth factor (EGF) Epithelial to mesenchymal transition (EMT) Fibroblast growth factor (FGF) Glioblastoma multiforme (GBM) Glioblastoma stem-like cell (GSC) Iso-dehydrogenase 1 (IDH1) Neural progenitor cell (NPC) Neural stem cell (NSC) O-6-methylguanine-DNA-methyltransferase (MGMT) Temozolomide (TMZ)11/2014; DOI:10.14343/JCSCR.2014.2e1007
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ABSTRACT: Within 2 hours of infection by Theileria annulata sporozoites bovine macrophages display a 2-4-fold increase in transcription of HIF-1α. Twenty hours post-invasion sporozoites develop into multi-nucleated macroschizonts that transform the infected macrophage into an immortalized, permanently proliferating, hyper-invasive and disease causing leukemia-like cell. Once immortalized Theileria-infected leukocytes can be propagated as cell lines and even though cultivated under normoxic conditions, both infected B cells and macrophages display sustained activation of HIF-1α. Attenuated macrophages used as live vaccines against tropical theileriosis also display HIF-1α activation even though they have lost their tumorigenic phenotype. Here, we review data that ascribes HIF-1α activation to the proliferation status of the infected leukocyte and discuss the possibility that Theileria may have lost its ability to render its host macrophage virulent due to continuous parasite replication in a high ROS environment. We propose a model, where uninfected macrophages have low levels of H2O2 output, whereas virulent infected macrophages produce high amounts of H2O2. Further increase in H2O2 output leads to dampening of infected macrophage virulence, a characteristic of disease-resistant macrophages. At the same time exposure to H2O2 sustains HIF-1α that induces the switch from mitochondrial oxidative phosphorylation to Warburg glycolysis, a metabolic shift that underpins uncontrolled infected macrophage proliferation. We propose that as macroschizonts develop into merozoites and infected macrophage proliferation arrests, HIF-1α levels will decrease and glycolysis will switch back from Warburg to oxidative glycolysis. As Theileria infection transforms its host leukocyte into an aggressive leukemic-like cell we propose that manipulating ROS levels, HIF-1α induction and oxidative over Warburg glycolysis could contribute to improved disease control. Finally, as excess amounts of H2O2 drive virulent Theileria-infected macrophages towards attenuation it highlights how infection-induced pathology and redox balance are intimately linked.Cellular Microbiology 01/2015; DOI:10.1111/cmi.12421 · 4.82 Impact Factor