Pulmonary, cardiac and renal syndrome.
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Adrenal Cortex Hormones↗
Biomedical subjects
J M Gleadle
Publications and source records attributed to J M Gleadle.
Regulation of gene expression by oxygen levels in mammalian cells.
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Animals↗
Role of the hypoxia sensing system, acidity and reproductive hormones in the variability of vascular endothelial growth factor induction in human breast carcinoma cell lines.
Vascular endothelial growth factor (VEGF) is a potent angiogenic factor implicated in many pathological processes. We investigated the regulation of 4 alternatively spliced isoforms (121, 165, 189 and 206 amino acids) by hypoxia, hypoglycemia, acidity, female reproductive hormones and vitamin D in breast carcinoma cell lines representing different tumor phenotypes. There was a 17-fold difference in total VEGF mRNA expression across the cell lines. The isoform expression, 121 > 165 > 189, was unchanged by different culture conditions. Hypoxia was the most potent stimulus, and the cell lines demonstrated a 1.4- to 6.9-fold range of VEGF induction, maintained when other hypoxically regulated genes (phosphoglycerate kinase 1 and glucose transporter 1) and a HIF-1-dependent reporter gene were examined. The relative inducibility of the genes was maintained in each cell line, but basal expression was independent of -fold induction. VEGF expression decreased under acidic conditions in 2 cell lines, but the hypoxia stimulus remained effective under acidic conditions. Hypoglycemia, female reproductive hormones and vitamin D exerted no effect on expression, nor did inhibitors of mutant ras. Our results show that VEGF expression varies widely between cell lines and that capacity to respond to hypoxia is also cell specific, relating mostly to the hypoxic sensing of the cell and the signal transduction mechanism. Such characteristics, if maintained in vivo, have implications for the angiogenic potential of different tumor cells under normal and hypoxic conditions.
Breast Neoplasms↗
Hypoxia and the regulation of gene expression.
The optimal delivery of oxygen to tissues is essential both to ensure adequate energy provision and to avoid the toxic effects of higher oxygen concentrations. For this to occur, organisms must be able to sense oxygen and respond to changes in oxygen tension by altering gene expression. The analysis of the regulation of erythropoiesis has provided important insights into the mechanisms of oxygen-regulated gene expression. These mechanisms have a role in the regulation of many genes, in many cell types and appear to be of relevance to many common pathologies in which disturbances of oxygen supply are central.
Animals↗
Hypoxia-inducible factor-1 modulates gene expression in solid tumors and influences both angiogenesis and tumor growth.
Recent studies of tissue culture cells have defined a widespread system of oxygen-regulated gene expression based on the activation of a heterodimeric transcription factor termed hypoxia-inducible factor-1 (HIF-1). To determine whether the HIF-1 transcriptional response is activated within solid tumors and to define the consequences, we have studied tumor xenografts of a set of hepatoma (Hepa-1) cells that are wild type (wt), deficient (c4), and revertant (Rc4) for an obligatory component of the HIF-1 heterodimer, HIF-1beta. Because HIF-1beta is also essential for the xenobiotic response (in which it is termed the aryl hydrocarbon receptor nuclear translocator), we also studied c31 cells, which have a different defect in the xenobiotic response and form the HIF-1 complex normally. Two genes that show different degrees of HIF-1-dependent hypoxia-inducible expression in cell culture were selected for analysis-the glucose transporter, GLUT3, and vascular endothelial growth factor (VEGF). In situ hybridization showed intense focal induction of gene expression in tumors derived from wt, Rc4, and c31 cells, which was reduced (VEGF) or not seen (GLUT3) in those derived from c4 cells. In association with these changes, tumors of c4 cells had reduced vascularity and grew more slowly. These findings show that HIF-1 activation occurs in hypoxic regions of tumors and demonstrate a major influence on gene expression, tumor angiogenesis, and growth.
Animals↗
Activation of hypoxia-inducible factor-1; definition of regulatory domains within the alpha subunit.
Hypoxia-inducible factor-1 (HIF-1), a heterodimeric DNA binding complex composed of two basic-helix-loop-helix Per-AHR-ARNT-Sim proteins (HIF-1alpha and -1beta), is a key component of a widely operative transcriptional response activated by hypoxia, cobaltous ions, and iron chelation. To identify regions of HIF-1 subunits responsible for oxygen-regulated activity, we constructed chimeric genes in which portions of coding sequence from HIF-1 genes were either linked to a heterologous DNA binding domain or encoded between such a DNA binding domain and a constitutive activation domain. Sequences from HIF-1alpha but not HIF-1beta conferred oxygen-regulated activity. Two minimal domains within HIF-1alpha (amino acids 549-582 and amino acids 775-826) were defined by deletional analysis, each of which could act independently to convey inducible responses. Both these regions confer transcriptional activation, and in both cases adjacent sequences appeared functionally repressive in transactivation assays. The inducible operation of the first domain, but not the second, involved major changes in the level of the activator fusion protein in transfected cells, inclusion of this sequence being associated with a marked reduction of expressed protein level in normoxic cells, which was relieved by stimulation with hypoxia, cobaltous ions, or iron chelation. These results lead us to propose a dual mechanism of activation in which the operation of an inducible activation domain is amplified by regulation of transcription factor abundance, most likely occurring through changes in protein stability.
Amino Acid Sequence↗
Induction of hypoxia-inducible factor-1, erythropoietin, vascular endothelial growth factor, and glucose transporter-1 by hypoxia: evidence against a regulatory role for Src kinase.
The induction by hypoxia of genes such as erythropoietin, vascular endothelial growth factor (VEGF), and glucose transporter-1 (Glut-1) is mediated in part by a transcriptional complex termed hypoxia-inducible factor-1 (HIF-1). Several lines of evidence have implicated protein phosphorylation in the mechanism of activation of HIF-1 by hypoxia. Recent reports have described the activation of the tyrosine kinase src by severe hypoxia, and a role in the induction of VEGF by severe hypoxia has been proposed. This led us to examine whether src and related kinases operated more widely in the hypoxic induction of HIF-1 and HIF-1-dependent genes regulated by hypoxia. Measurements of src kinase activity in cells exposed to varying severities of hypoxia showed activation by severe hypoxia (0.1% oxygen or catalyst induced anoxia), but not 1% oxygen. This contrasted with the marked induction of HIF-1 by exposure to 1% oxygen. Manipulations of src activity were produced by transient and stable transfection of Hep3B cells. Despite substantial changes in src activity, no alteration was seen in the normoxic or hypoxic expression of erythropoietin, VEGF, or Glut-1, or in the regulation of HIF-1-dependent reporter genes inducible by hypoxia. Similarly, we found that the expression of these genes in src- or c-src kinase-deficient cells did not differ from wild-type cells at either 1% oxygen or more severe hypoxia. These results indicate that src is not critical for the hypoxic induction of HIF-1, erythropoietin, VEGF, or Glut-1.
Animals↗
Oxygen regulated gene expression: erythropoietin as a model system.
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Animals↗
Hypoxia response elements.
Hypoxia-inducible factor-1 (HIF-1) has been shown to mediate the transcriptional activation of its target genes in response to oxygen concentration, most likely via a pathway involving a specific oxygen sensor. Molecular cloning of HIF-1 has shown that this widely expressed, DNA binding transcription factor is a heterodimer of two proteins, HIF-1 alpha and HIF-1 beta. A major control of HIF-1 activity by oxygen tension is achieved by changes in the level of the HIF-1 alpha subunit, which complexes with the constitutively expressed HIF-1 beta subunit. Such changes in HIF-1 alpha abundance occur via regulated stability, probably involving proteolysis, rather than at the level of transcription or translation. Further analysis has shown the existence of two separate regulatory domains in the C-terminus of the alpha subunit. Thus, a mechanism of oxygen-regulated HIF-1 activation is proposed, which involves the operation of one inducible domain being amplified by changes in protein level conferred by a second regulatory domain. Evidence for a critical role of HIF-1 in the response of diverse target genes involved in cellular growth and metabolism comes from studies on cultured, mutant mouse cells that lack a functional HIF-1 beta subunit. Furthermore, studies on tumor xenografts derived from the mutant and wild-type cells show that HIF-1 is activated in vivo, and has major effects on gene expression in response to tumor hypoxia. Thus, HIF-1 is a critical component of the oxygen-signaling pathway, and is a prime candidate regulator molecule for the role of coordinating vascular oxygen supply with cellular growth and energy metabolism.
Animals↗
The role of the aryl hydrocarbon receptor nuclear translocator (ARNT) in hypoxic induction of gene expression. Studies in ARNT-deficient cells.
Hypoxia-inducible factor-1 (HIF-1), a DNA-binding complex implicated in the regulation of gene expression by oxygen, has been shown to consist of a heterodimer of two basic helix-loop-helix Per-AHR-ARNT-Sim (PAS) proteins, HIF-1alpha, and HIF-1beta. One partner, HIF-1beta, had been recognized previously as the aryl hydrocarbon receptor nuclear translocator (ARNT), an essential component of the xenobiotic response. In the present work, ARNT-deficient mutant cells, originally derived from the mouse hepatoma line Hepa1c1c7, have been used to analyze the role of ARNT/HIF-1beta in oxygen-regulated gene expression. Two stimuli were examined: hypoxia itself and desferrioxamine, an iron-chelating agent that also activates HIF-1. Induction of the DNA binding and transcriptional activity of HIF-1 was absent in the mutant cells, indicating an essential role for ARNT/HIF-1beta. Analysis of deleted ARNT/HIF-1beta genes indicated that the basic, helix-loop-helix, and PAS domains, but not the amino or carboxyl termini, were necessary for function in the response to hypoxia. Comparison of gene expression in wild type and mutant cells demonstrated the critical importance of ARNT/HIF-1beta in the hypoxic induction of a wide variety of genes. Nevertheless, for some genes a reduced response to hypoxia and desferrioxamine persisted in these mutant cells, clearly distinguishing ARNT/HIF-1beta-dependent and ARNT/HIF-1beta-independent mechanisms of gene activation by both these stimuli.
Animals↗
Isoenzyme-specific regulation of genes involved in energy metabolism by hypoxia: similarities with the regulation of erythropoietin.
Recent studies have indicated that regulatory mechanisms underlying the oxygen-dependent expression of the haematopoietic growth factor erythropoietin are widely operative in non-erythropoietin-producing cells and are involved in the regulation of other genes. An important characteristic of this system is that the inducible response to hypoxia is mimicked by exposure to particular transition metals such as cobaltous ions, and by iron chelation. We have investigated the extent of operation of this system in the regulation of a range of genes concerned with energy metabolism. The effects of hypoxia (1% oxygen), cobaltous ions and desferrioxamine on gene expression in tissue-culture cells was studied using RNase protection assays. Hypoxia induced the expression of glucose transporters in an isoform-specific manner; GLUT-1 and GLUT-3 were induced by hypoxia, whereas expression of GLUT-2 was decreased. Isoenzyme-specific regulation by hypoxia was also observed for genes encoding phosphofructokinase, aldolase and lactate dehydrogenase. For all of these genes, responses to cobaltous ions and desferrioxamine correlated in both direction and magnitude with the response to hypoxia. In contrast, a reduction in mitochondrial transcripts was observed in hypoxia, but these changes were not mimicked by either cobaltous ions or desferrioxamine. These findings indicate that similarities with erythropoietin regulation extend to the oxygen-dependent regulation of genes encoding glucose transporters and glycolytic enzymes but not to the regulation of mitochondrial transcripts, and they show that in glucose metabolism regulation by this system is isoenzyme- or isoform-specific.
Base Sequence↗
Diphenylene iodonium inhibits the induction of erythropoietin and other mammalian genes by hypoxia. Implications for the mechanism of oxygen sensing.
Recent studies on the induction of erythropoietin gene expression by hypoxia have indicated that erythropoietin forms part of a widely operative system of gene regulation by oxygen. Similar responses to hypoxia, cobaltous ions and desferrioxamine have indicated that the action of these agents is closely connected with the mechanism of oxygen sensing. To consider further the mechanisms underlying these responses, the effect of iodonium compounds was tested on five genes which show oxygen-regulated expression; erythropoietin, vascular endothelial growth factor (VEGF), lactate dehydrogenase-A (LDH-A), glucose transporter-1 (GLUT-1) and placental growth factor (PLGF). In each case, the response to hypoxia was specifically inhibited by low doses of diphenylene iodonium (Ph1I+). This occurred irrespective of whether the hypoxic response was induction of gene expression (erythropoietin, vascular endothelial growth factor, lactate dehydrogenase-A, glucose transporter-1) or inhibition of gene expression (PLGF). In contrast, the induction of gene expression by cobaltous ions or desferrioxamine was not inhibited by Ph2I+. The differential action of Ph2I+ on the response to hypoxia versus the response to cobaltous ions or desferrioxamine must reflect a difference in the mechanism of action of these stimuli, which will require accommodation in any model of the oxygen-sensing mechanism. Based on the known properties of Ph2I+, the implication of these findings is that the mechanism of oxygen sensing most probably involves the operation of a flavoprotein oxidoreductase.
Base Sequence↗
Regulation of the erythropoietin gene.
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Animals↗
Regulation of angiogenic growth factor expression by hypoxia, transition metals, and chelating agents.
Recent work has indicated that oxygen-sensing mechanism(s) resembling those controlling erythropoietin production operate in many non-erythropoietin-producing cells. To pursue the implication that such a system might control other genes, we studied oxygen-regulated expression of mRNAs for vascular endothelial growth factor, platelet-derived growth factor (PDGF) A and B chains, placental growth factor (PLGF), and transforming growth factor in four different cell lines and compared the characteristics with those of erythropoietin regulation. Oxygen-regulated expression was demonstrated for each gene in at least one cell type. However, the response to hypoxia (1% oxygen) varied markedly, ranging from a 13-fold increase (PDGF-B in Hep G2 cells) to a 2-fold decrease (PLGF in the trophoblastic line BeWo). For each gene/cell combination, both the magnitude and direction of the response to hypoxia were mimicked by exposure to cobaltous ions or two different iron-chelating agents, desferrioxamine and hydroxypyridinones. These similarities with established characteristics of erythropoietin regulation indicate that a similar mechanism of oxygen sensing is operating on a variety of vascular growth factors, and they suggest that chelatable iron is closely involved in the mechanism.
Angiogenesis Inducing Agents↗