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H F Bunn

Publications and source records attributed to H F Bunn.

At least 19 recordsLinked to original sources
PubMed1999

Inhibition of hypoxia-inducible factor 1 activation by carbon monoxide and nitric oxide. Implications for oxygen sensing and signaling.

It has been proposed that cells sense hypoxia by a heme protein, which transmits a signal that activates the heterodimeric transcription factor hypoxia-inducible factor 1 (HIF-1), thereby inducing a number of physiologically relevant genes such as erythropoietin (Epo). We have investigated the mechanism by which two heme-binding ligands, carbon monoxide and nitric oxide, affect oxygen sensing and signaling. Two concentrations of CO (10 and 80%) suppressed the activation of HIF-1 and induction of Epo mRNA by hypoxia in a dose-dependent manner. In contrast, CO had no effect on the induction of HIF-1 activity and Epo expression by either cobalt chloride or the iron chelator desferrioxamine. The affinity of CO for the putative sensor was much lower than that of oxygen (Haldane coefficient, approximately 0.5). Parallel experiments were done with 100 microM sodium nitroprusside, a nitric oxide donor. Both NO and CO inhibited HIF-1 DNA binding by abrogating hypoxia-induced accumulation of HIF-1alpha protein. Moreover, both NO and CO specifically targeted the internal oxygen-dependent degradation domain of HIF-1alpha, and also repressed the C-terminal transactivation domain of HIF-1alpha. Thus, NO and CO act proximally, presumably as heme ligands binding to the oxygen sensor, whereas desferrioxamine and perhaps cobalt appear to act at a site downstream.

Aryl Hydrocarbon Receptor Nuclear Translocator↗
PubMed1998

Regulation of hypoxia-inducible factor 1alpha is mediated by an O2-dependent degradation domain via the ubiquitin-proteasome pathway.

Hypoxia induces a group of physiologically important genes such as erythropoietin and vascular endothelial growth factor. These genes are transcriptionally up-regulated by hypoxia-inducible factor 1 (HIF-1), a global regulator that belongs to the basic helix-loop-helix PAS family. Although HIF-1 is a heterodimer composed of alpha and beta subunits, its activity is primarily determined by hypoxia-induced stabilization of HIF-1alpha, which is otherwise rapidly degraded in oxygenated cells. We report the identification of an oxygen-dependent degradation (ODD) domain within HIF-1alpha that controls its degradation by the ubiquitin-proteasome pathway. The ODD domain consists of approximately 200 amino acid residues, located in the central region of HIF-1alpha. Because portions of the domain independently confer degradation of HIF-1alpha, deletion of this entire region is required to give rise to a stable HIF-1alpha, capable of heterodimerization, DNA-binding, and transactivation in the absence of hypoxic signaling. Conversely, the ODD domain alone confers oxygen-dependent instability when fused to a stable protein, Gal4. Hence, the ODD domain plays a pivotal role for regulating HIF-1 activity and thereby may provide a means of controlling gene expression by changes in oxygen tension.

Animals↗
PubMed1998

Homodimerization restores biological activity to an inactive erythropoietin mutant.

Erythropoietin (Epo) is believed to transduce a signal by bringing two Epo receptors into close proximity, enabling cross-phosphorylation. We compared monomeric Epos with homodimers in which two Epo monomers are linked by polyglycine. Monomeric Epo mutant R103A is unable to support Epo-dependent cell growth or trigger Janus kinase 2 and STAT5 activation, even at concentrations greater than 7,000 times that sufficient for wild-type Epo activity. In contrast, R103A homodimer induces proliferation and transduces signal at concentrations similar to that of wild-type Epo monomer and homodimer. These experiments show that two discrete domains on Epo are required for receptor binding and activation. Our results also suggest that the EpoR can be dimerized by different forms and sizes of molecules, as long as two recognition motifs are provided in the same molecule. Design of other dimeric molecules may enhance our understanding of cytokine specificity and signal transduction.

Animals↗
PubMed1998

Regulation of transcription by hypoxia requires a multiprotein complex that includes hypoxia-inducible factor 1, an adjacent transcription factor, and p300/CREB binding protein.

Molecular adaptation to hypoxia depends on the binding of hypoxia-inducible factor 1 (HIF-1) to cognate response elements in oxygen-regulated genes. In addition, adjacent sequences are required for hypoxia-inducible transcription. To investigate the mechanism of interaction between these cis-acting sequences, the multiprotein complex binding to the lactate dehydrogenase A (LDH-A) promoter was characterized. The involvement of HIF-1, CREB-1/ATF-1, and p300/CREB binding protein (CBP) was demonstrated by techniques documenting in vitro binding, in combination with transient transfections that test the in vivo functional importance of each protein. In both the LDH-A promoter and the erythropoietin 3' enhancer, formation of multiprotein complexes was analyzed by using biotinylated probes encompassing functionally critical cis-acting sequences. Strong binding of p300/CBP required interactions with multiple DNA binding proteins. Thus, the necessity of transcription factor binding sites adjacent to a HIF-1 site for hypoxically inducible transcription may be due to the requirement of p300 to interact with multiple transcription factors for high-affinity binding and activation of transcription. Since it has been found to interact with a wide range of transcription factors, p300 is likely to play a similar role in other genes, mediating interactions between DNA binding proteins, thereby activating stimulus-specific and tissue-specific gene transcription.

Activating Transcription Factor 1↗
PubMed1998

Erythropoietin: a model system for studying oxygen-dependent gene regulation.

The physiological regulation of the red cell mass depends upon enhanced transcription of the erythropoietin (Epo) gene in response to hypoxia. Studies of Epo gene expression have been useful in investigating the mechanism by which cells and tissues sense hypoxia and respond with biologically appropriate alterations in gene expression. It is likely that oxygen sensing involves a heme protein in which cobalt and nickel can substitute for iron in the porphyrin ring. Indirect evidence suggests that the sensor is present in all cells and is a multi-subunit assembly containing an NAD(P)H oxidase capable of generating peroxide and reactive oxygen intermediates, which serve as signaling molecules. The up-regulation of Epo gene transcription by hypoxia is mediated by at least two known DNA-binding transcription factors, hypoxia-inducible factor 1 (HIF-1) and hepatic nuclear factor 4 (HNF-4), which bind to cognate response elements in a critical 3' enhancer approximately 50 bp in length. HIF-1 binding is induced by hypoxia as well as by cobalt. The activation of HIF-1 by hypoxia depends upon the selective protection of its alpha subunit from ubiquitin-dependent proteolysis by means of a mechanism that involves redox chemistry and perhaps phosphorylation. HNF-4 is an orphan nuclear receptor that is constitutively expressed in kidney and liver and which cooperates with HIF-1 to give maximal hypoxic induction. In hypoxic cells, p300 or a related family member forms a macromolecular assembly with HIF-1 and HNF-4, enabling transduction from the Epo 3' enhancer to the apparatus on the promoter responsible for the initiation of transcription.

Animals↗
PubMed1997

Erythropoietin gene regulation depends on heme-dependent oxygen sensing and assembly of interacting transcription factors.

Studies on erythropoietin (Epo) gene expression have been useful in investigating the mechanism by which cells and tissues sense hypoxia. Both in vivo and in Hep3B cells. Epo production is induced not only by hypoxia but also by certain transition metal (cobalt and nickel) and by iron chelation. When Hep3B cells were incubated in an iron deficient medium, Epo mRNA expression was enhanced fourfold compared to Hep3B cells in iron enriched medium. Epo induction by cobalt was inversely related to iron concentration in the medium, indicating competition between the two metals. Under hyperbaric oxygen, cobalt induction of erythropoietin mRNA was modestly suppressed while nickel induction was markedly enhanced. These recent observations support the proposal that the oxygen sensor is a heme protein in which cobalt and nickel can substitute for iron in the porphyrin ring. The up-regulation of Epo gene transcription by hypoxia depends on at least two known DNA binding transcription factors, HIF-1 and HNF-4, which bind to cognate response elements in a critical approximately 50 bp 3' enhancer. Hypoxia induces HIF-1 binding. HNF-4, an orphan nuclear receptor constitutively expressed in kidney and liver, binds downstream of HIF-1 and cooperates with HIF-1, contributing importantly to high level and perhaps tissue specific expression. The C-terminal activation domain of HNF-4 binds to the beta subunit of HIF-1. The C-terminal portion of the alpha subunit of HIF-1 binds specifically to p300, a general transcriptional activator. Hypoxic induction of the endogenous Epo gene in Hep3B cells as well as an Epo-reporter gene was fully inhibited by E1A, an adenovirus protein that binds to and inactivates p300, but only slightly by a mutant E1A that fails to bind to p300. Moreover, overexpression of p300 enhanced hypoxic induction. Thus, it is likely that in hypoxic cells, p300 or a related family member plays a critical role in forming a macromolecular assembly with HIF-1 and HNF-4, enabling transduction from the Epo 3' enhancer to the apparatus on the promoter responsible for the initiation of transcription.

Basic Helix-Loop-Helix Leucine Zipper Transcriptio↗
PubMed1996

Activation of hypoxia-inducible transcription factor depends primarily upon redox-sensitive stabilization of its alpha subunit.

Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric transcription factor that is critical for hypoxic induction of a number of physiologically important genes. We present evidence that regulation of HIF-1 activity is primarily determined by the stability of the HIF-1alpha protein. Both HIF-1alpha and HIF-1beta mRNAs were constitutively expressed in HeLa and Hep3B cells with no significant induction by hypoxia. However, the HIF-1alpha protein was barely detectable in normoxic cells, even when HIF-1alpha was overexpressed, but was highly induced in hypoxic cells, whereas HIF-1beta protein levels remained constant, regardless of pO2. Hypoxia-induced HIF-1 binding as well as the HIF-1alpha protein were rapidly and drastically decreased in vivo following an abrupt increase to normal oxygen tension. Moreover, short pre-exposure of cells to hydrogen peroxide selectively prevented hypoxia-induced HIF-1 binding via blocking accumulation of HIF-1alpha protein, whereas treatment of hypoxic cell extracts with H2O2 had no effect on HIF-1 binding. These observations suggest that an intact redox-dependent signaling pathway is required for destablization of the HIF-1alpha protein. In hypoxic cell extracts, HIF-1 DNA binding was reversibly abolished by sulfhydryl oxidation. Furthermore, the addition of reduced thioredoxin to cell extracts enhanced HIF-1 DNA binding. Consistent with these results, overexpression of thioredoxin and Ref-1 significantly potentiated hypoxia-induced expression of a reporter construct containing the wild-type HIF-1 binding site. These experiments indicate that activation of HIF-1 involves redox-dependent stabilization of HIF-1alpha protein.

Blotting, Western↗
PubMed1996

An essential role for p300/CBP in the cellular response to hypoxia.

p300 and CBP are homologous transcription adapters targeted by the E1A oncoprotein. They participate in numerous biological processes, including cell cycle arrest, differentiation, and transcription activation. p300 and/or CBP (p300/CBP) also coactivate CREB. How they participate in these processes is not yet known. In a search for specific p300 binding proteins, we have cloned the intact cDNA for HIF-1 alpha. This transcription factor mediates hypoxic induction of genes encoding certain glycolytic enzymes, erythropoietin (Epo), and vascular endothelial growth factor. Hypoxic conditions lead to the formation of a DNA binding complex containing both HIF-1 alpha and p300/CBP. Hypoxia-induced transcription from the Epo promoter was specifically enhanced by ectopic p300 and inhibited by E1A binding to p300/CBP. Hypoxia-induced VEGF and Epo mRNA synthesis were similarly inhibited by E1A. Hence, p300/CBP-HIF complexes participate in the induction of hypoxia-responsive genes, including one (vascular endothelial growth factor) that plays a major role in tumor angiogenesis. Paradoxically, these data, to our knowledge for the first time, suggest that p300/ CBP are active in both transformation suppression and tumor development.

Adenovirus E1A Proteins↗
PubMed1996

Effects of transition metals on the expression of the erythropoietin gene: further evidence that the oxygen sensor is a heme protein.

Both in vivo and in Hep3B cells, expression of the erythropoietin gene is induced by hypoxia as well as by certain transition metals (cobalt and nickel) and by iron chelation. When Hep3B cells were incubated in an iron deficient medium, Epo mRNA expression was enhanced 4-fold compared to Hep3B cells in iron enriched medium. The increased Epo expression in iron deficient medium was abolished when Fe2-transferrin complex was added. Epo induction by cobalt was also affected by iron concentration. In iron enriched medium, erythropoietin expression in Hep3B cells was maximally induced at CoCl2 concentrations between 100 to 200 microM. In contrast, in iron poor medium, a high level of induction was obtained at a CoCl2 concentration of only 50 microM, indicating competition between iron and cobalt. Under hyperbaric oxygen, cobalt induction of erythropoietin mRNA was modestly suppressed while nickel induction was markedly enhanced. These observations support the proposal that the oxygen sensor is a heme protein in which cobalt and nickel can substitute for iron in the porphyrin ring.

Carcinoma, Hepatocellular↗
PubMed1996

Oxygen sensing and molecular adaptation to hypoxia.

This review focuses on the molecular stratagems utilized by bacteria, yeast, and mammals in their adaptation to hypoxia. Among this broad range of organisms, changes in oxygen tension appear to be sensed by heme proteins, with subsequent transfer of electrons along a signal transduction pathway which may depend on reactive oxygen species. These heme-based sensors are generally two-domain proteins. Some are hemokinases, while others are flavohemoproteins [flavohemoglobins and NAD(P)H oxidases]. Hypoxia-dependent kinase activation of transcription factors in nitrogen-fixing bacteria bears a striking analogy to the phosphorylation of hypoxia inducible factor-1 (HIF-1) in mammalian cells. Moreover, redox chemistry appears to play a critical role both in the trans-activation of oxygen-responsive genes in unicellular organisms as well as in the activation of HIF-1. In yeast and bacteria, regulatory operons coordinate expression of genes responsible for adaptive responses to hypoxia and hyperoxia. Similarly, in mammals, combinatorial interactions of HIF-1 with other identified transcription factors are required for the hypoxic induction of physiologically important genes.

Animals↗
PubMed1995

Use of a marked erythropoietin gene for investigation of its cis-acting elements.

To examine the function of conserved noncoding regions in the erythropoietin (Epo) gene, we have prepared clones and pools of Hep3B cells stably transfected with a marked 4.1-kilobase Epo gene and deletions thereof. The marked transcripts had single base substitutions at three sites in the coding portion of Exon 5, enabling them to be distinguished from endogenous Epo mRNA by ribonuclease protection and competitive polymerase chain reaction. The basal expression and hypoxic induction of the marked Epo gene that had no deletions were indistinguishable from that of the endogenous Epo gene. Likewise, deletion of conserved intervening sequence 1 had minimal effect on hypoxic induction. In contrast, a 3'-deletion that included the conserved 3'-enhancer element resulted in a substantial, but not complete, suppression of hypoxic induction while a 3'-deletion downstream of the enhancer resulted in enhancement. A 188-base pair deletion of a conserved 3'-untranslated region in Exon 5 had minimal effect on hypoxic induction. However, the truncated Epo mRNA had a markedly prolonged half-life (15 h) in comparison to the endogenous Epo mRNA (2.0 h) or the marked full-length Epo mRNA (2.1 h). Further deletions in the 3'-UTR showed that a relatively small region of approximately 50 bases is responsible for the relatively rapid turnover of Epo mRNA. These experiments provide information on cis-acting elements of the Epo gene that cannot be obtained from conventional reporter gene transfection experiments.

Base Sequence↗
PubMed1995

Comments on the physics and chemistry of trehalose as a storage medium for hemoglobin-based blood substitutes: "from Kramers Theory to the Battlefield".

A glass of the naturally-occurring sugar trehalose may be a suitable medium for the storage of hemoglobin-based blood substitutes. Trehalose has many or possibly all of the properties required for this purpose, including solubilization of hemoglobin to a very high concentration, lack of toxicity, slowing of oxidation to the non-oxygen binding methemoglobin, stability at room temperature and above, and ease of transport. It should also be possible to prepare hemoglobin extremely rapidly for injection into the circulation in situations where blood replacement is required immediately, as in a domestic emergency room or on the battlefield. These practical considerations are briefly discussed, as well as the theoretical reasons for slowing of chemical reactions in the glassy state.

Blood Substitutes↗
PubMed1995

The role of hemoglobin based blood substitutes in transfusion medicine.

A cell-free oxygen transporting blood substitute would obviate many of the current concerns about conventional red cell transfusion therapy. Moreover, a stable oxygen-carrying solution could have benefits and applications not possible with red cell transfusions, such as the treatment of acute hypovolemic shock in acute care settings, the treatment of patients such as Jehovah's Witnesses who refuse blood transfusions, the priming of blood oxygenation pumps, ex vivo organ perfusion prior to transplantation, and in vivo perfusion in order to enhance sensitivity to radiation therapy. Among potential blood substitutes that transport oxygen, attention has focused on perfluorocarbons and a variety of hemoglobin preparations, either in free solution or encapsulated into lipid vesicles. In the design and production of hemoglobin solutions the following criteria must be met: low toxicity and antigenicity; efficacy as a plasma expander; prolonged survival in the circulation; adequate oxygen carrying capability and efficient oxygen unloading to tissues; long shelf life. Extensive preclinical testing and recent clinical trials have been performed on human and bovine hemoglobin chemically crosslinked to present rapid leakage of hemoglobin through the kidneys. Bovine hemoglobin has intrinsically low oxygen affinity simulating that of human hemoglobin in red cells. An alternative and attractive strategy is the production of human hemoglobin in E. Coli, thus enabling appropriate genetic mutations to optimize function. These include creation of peptide linkers to enhance plasma survival and amino acid replacements that permit a finely regulated lowering of oxygen affinity.

Animals↗
PubMed1995

Regulation of erythropoietin gene expression.

The study of erythropoietin gene expression provides a paradigm for understanding gene regulation in response to hypoxia. The sensor for detecting alterations in oxygen tension appears to be a heme protein. Ongoing transcription and protein synthesis are necessary for hypoxic induction of erythropoietin messenger RNA. In the past few years, considerable progress has been made in the identification and characterization of cis-acting elements and trans-acting factors that contribute to erythropoietin gene expression. The erythropoietin promoter and 3' enhancer function synergistically in response to hypoxia. Whereas hypoxia-inducible factor 1 specifically binds to the 3' enhancer conferring hypoxic induction, hepatic nuclear factor 4 interacts with the promoter as well as the 3' enhancer for stimulus- and tissue-specific induction of the erythropoietin gene. In addition, a segment in the 3' untranslated region contributes to the relatively rapid turnover of erythropoietin messenger RNA.

Animals↗