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Peter J Ratcliffe

Publications and source records attributed to Peter J Ratcliffe.

48 records · Page 3Linked to original sources
PubMed2002

Hypoxia-inducible factor (HIF) asparagine hydroxylase is identical to factor inhibiting HIF (FIH) and is related to the cupin structural family.

Activity of the hypoxia-inducible factor (HIF) complex is controlled by oxygen-dependent hydroxylation of prolyl and asparaginyl residues. Hydroxylation of specific prolyl residues by 2-oxoglutarate (2-OG)-dependent oxygenases mediates ubiquitinylation and proteasomal destruction of HIF-alpha. Hydroxylation of an asparagine residue in the C-terminal transactivation domain (CAD) of HIF-alpha abrogates interaction with p300, preventing transcriptional activation. Yeast two-hybrid assays recently identified factor inhibiting HIF (FIH) as a protein that associates with the CAD region of HIF-alpha. Since FIH contains certain motifs present in iron- and 2-OG-dependent oxygenases we investigated whether FIH was the HIF asparaginyl hydroxylase. Assays using recombinant FIH and HIF-alpha fragments revealed that FIH is the enzyme that hydroxylates the CAD asparagine residue, that the activity is directly inhibited by cobalt(II) and limited by hypoxia, and that the oxygen in the alcohol of the hydroxyasparagine residue is directly derived from dioxygen. Sequence analyses involving FIH link the 2-OG oxygenases with members of the cupin superfamily, including Zn(II)-utilizing phosphomannose isomerase, revealing structural and evolutionary links between these metal-binding proteins that share common motifs.

Amino Acid Sequence↗
PubMed2002

Expression of hypoxia-inducible factors in human renal cancer: relationship to angiogenesis and to the von Hippel-Lindau gene mutation.

The von Hippel-Lindau tumor suppressor protein acts as the substrate recognition component of a ubiquitin E3 ligase that targets hypoxia-inducible factor (HIF)-alpha subunits for proteolysis. Stabilization of HIF-alpha subunits has been described in VHL-defective cell lines, leading to HIF activation and up-regulation of hypoxia-inducible mRNAs. Mutations of the von Hippel-Lindau tumor suppressor protein are found in most clear cell renal cell carcinomas (CC-RCCs) but not other renal tumors, raising a question about the importance of activation of the HIF pathway in CC-RCC development. To address this question, we have examined the expression of HIF-alpha subunits in 45 primary renal tumors and related this to tumor subtype, the presence of VHL mutations, and measures of angiogenesis. We show that HIF-alpha is up-regulated in the majority of CC-RCCs, and that the pattern of expression is biased toward the HIF-2alpha isoform. Expression of HIF-alpha proteins was associated significantly with up-regulation of VEGF mRNA and protein and increased microvessel density. Up-regulation of HIF-alpha in CC-RCC was found to involve increased mRNA as well as protein expression, suggesting that both VHL-dependent and VHL-independent mechanisms are involved. These results suggest that activation of the HIF pathway is functionally important in CC-RCC development and might provide a new therapeutic target.

Adenocarcinoma, Clear Cell↗
PubMed2002

Oxygen sensors and angiogenesis.

Local oxygen tension has a profound effect on the vasculature, which compensates vascular insufficiency through the induction of angiogenesis. An important mediator in this process is the hypoxia-inducible factor (HIF) complex, which is activated in hypoxic cells and increases transcription of a broad range of genes including angiogenic growth factors such as VEGF. HIF is primarily regulated through oxygen-dependent proteasomal destruction of the regulatory subunit, HIF-1 alpha or HIF-2 alpha. Regulation is through the modification of specific prolines in HIF- alpha chains which are hydroxylated by a recently identified family of enzymes which require molecular oxygen and 2-oxoglutarate as cosubstrates, and iron as a cofactor. Following modification HIF- alpha chains are captured by a ubiquitin ligase E3 complex containing the von Hippel-Lindau (VHL) tumour suppressor protein. The HIF prolyl hydroxylases (PHD enzymes) act as oxygen sensors regulating HIF, and hence angiogenesis. The PHD-HIF-VHL system provides a range of opportunities for therapeutic manipulation.

Animals↗
PubMed2002

Genotype at a promoter polymorphism of the interleukin-6 gene is associated with baseline levels of plasma C-reactive protein.

OBJECTIVE: Baseline concentrations of plasma C-reactive protein (CRP) are associated with coronary heart disease. Interleukin-6 (IL-6) regulates CRP gene expression; a promoter polymorphism (-174G/C) of the IL-6 gene has been shown to influence IL-6 transcription but the relationship between genotype at this polymorphism and circulating levels of inflammatory markers remains unclear. We hypothesised that plasma CRP would be a heritable phenotype that would be influenced by genotype at this polymorphism. METHODS: We measured baseline plasma CRP and determined genotypes at the -174G/C polymorphism of the IL-6 gene in 588 members of 98 nuclear families. The heritability of plasma CRP and the association of plasma CRP with genotype were determined using variance components methods. RESULTS: Baseline CRP levels were highly heritable (h(2)=0.39, P<0.0000001). Presence of the -174C allele was associated with higher baseline CRP levels, both in the whole population (P=0.01), and in the founders only (n=128, P=0.001). Family-based analyses confirmed the association (P=0.02) suggesting that it arises from chromosomal proximity or identity of the typed polymorphism with a genetic variant influencing baseline CRP levels. CONCLUSIONS: Baseline plasma CRP is a significantly heritable cardiovascular risk factor. Levels are associated with genotype at the -174G/C polymorphism of the IL-6 gene.

Adult↗
PubMed2002

HIF activation identifies early lesions in VHL kidneys: evidence for site-specific tumor suppressor function in the nephron.

Mutations in the von Hippel-Lindau (VHL) gene are associated with hereditary and sporadic clear cell renal carcinoma. VHL acts in a ubiquitin ligase complex regulating hypoxia-inducible factor-1 (HIF-1), but the link between this function and cancer development is unclear. Here we show that in the kidneys of patients with VHL disease, HIF activation is an early event occurring in morphologically normal single cells within the renal tubules. In comparison, dysplastic lesions, cystic lesions, and tumors showed evidence of additional mechanisms that amplify HIF activation. Detection of cells with constitutive HIF activation identified a large number of previously unrecognized foci of VHL inactivation. In proximal tubules these were almost entirely unicellular, whereas multicellular foci were almost exclusively seen in the distal nephron.

Adenocarcinoma, Clear Cell↗
PubMed2002

Expression of hypoxia-inducible factor-1alpha and -2alpha in hypoxic and ischemic rat kidneys.

Oxygen tensions in the kidney are heterogeneous, and their changes presumably play an important role in renal physiologic and pathophysiologic processes. A family of hypoxia-inducible transcription factors (HIF) have been identified as mediators of transcriptional responses to hypoxia, which include the regulation of erythropoietin, metabolic adaptation, vascular tone, and neoangiogenesis. In vitro, the oxygen-regulated subunits HIF-1alpha and -2alpha are expressed in inverse relationship to oxygen tensions in every cell line investigated to date. The characteristics and functional significance of the HIF response in vivo are largely unknown. High-amplification immunohistochemical analyses were used to study the expression of HIF-1alpha and -2alpha in kidneys of rats exposed to systemic hypoxia bleeding anemia, functional anemia (0.1% carbon monoxide), renal ischemia, or cobaltous chloride (which is known to mimic hypoxia). These treatments led to marked nuclear accumulation of HIF-1alpha and -2alpha in different renal cell populations. HIF-1alpha was mainly induced in tubular cells, including proximal segments with exposure to anemia/carbon monoxide, in distal segments with cobaltous chloride treatment, and in connecting tubules and collecting ducts with all stimuli. Staining for HIF-1alpha colocalized with inducible expression of the target genes heme oxygenase-1 and glucose transporter-1. HIF-2alpha was not expressed in tubular cells but was expressed in endothelial cells of a small subset of glomeruli and in peritubular endothelial cells and fibroblasts. The kidney demonstrates a marked potential for upregulation of HIF, but accumulation of HIF-1alpha and HIF-2alpha is selective with respect to cell type, kidney zone, and experimental conditions, with the expression patterns partly matching known oxygen profiles. The expression of HIF-2alpha in peritubular fibroblasts suggests a role in erythropoietin regulation.

Animals↗
PubMed2002

Delivery of erythropoietin by encapsulated myoblasts in a genetic model of severe anemia.

BACKGROUND: Existing animal models of anemia inadequately reflect the hematocrit usually present in chronic renal failure (CRF) patients and do not permit long-term treatment studies. The transgenic mouse strain 134.3LC (Epo-TAg(H)) displays a severe chronic anemia resembling that observed clinically during CRF, while displaying an active, normal life span. This phenotype makes it a particularly interesting mouse model for testing erythropoietin (Epo)-based gene transfer strategies. METHODS: Ex vivo gene therapy was employed to administer mouse Epo to homozygous anemic Epo-TAg(H) mice. Encapsulated C(2)C(12) myoblasts genetically engineered to secrete 163 IU mouse Epo/10(6) cells/day were subcutaneously transplanted on the dorsal flank of the mice. Efficacy of delivered Epo was monitored by weekly measurements of animal hematocrit. RESULTS: Most treated homozygous Epo-TAg(H) mice displayed only a transient rise in hematocrit before eventually decreasing to levels as low as 3%. Administering the immunosuppressor anti-CD4+ monoclonal antibody (mAb) to homozygous Epo-TAg(H) mice, beginning at the time of implantation, permitted a rise in hematocrit that remained stable at elevated levels in cases of continued immunosuppression. CONCLUSIONS: Mice having the T antigen insertion in both Epo alleles appeared to develop an immune response to the natural mouse Epo delivered by encapsulated cells. By preventing this reaction using immunosuppression, we demonstrate that encapsulated myoblasts can deliver therapeutic doses of mouse Epo systemically and restore hemopoiesis in a genetic model of severe anemia.

Anemia↗
PubMed2002

Control of the hypoxic response in Drosophila melanogaster by the basic helix-loop-helix PAS protein similar.

In mammalian systems, the heterodimeric basic helix-loop-helix (bHLH)-PAS transcription hypoxia-inducible factor (HIF) has emerged as the key regulator of responses to hypoxia. Here we define a homologous system in Drosophila melanogaster, and we characterize its activity in vivo during development. By using transcriptional reporters in developing transgenic flies, we show that hypoxia-inducible activity rises to a peak in late embryogenesis and is most pronounced in tracheal cells. We show that the bHLH-PAS proteins Similar (Sima) and Tango (Tgo) function as HIF-alpha and HIF-beta homologues, respectively, and demonstrate a conserved mode of regulation for Sima by oxygen. Sima protein, but not its mRNA, was upregulated in hypoxia. Time course experiments following pulsed ectopic expression demonstrated that Sima is stabilized in hypoxia and that degradation relies on a central domain encompassing amino acids 692 to 863. Continuous ectopic expression overrode Sima degradation, which remained cytoplasmic in normoxia, and translocated to the nucleus only in hypoxia, revealing a second oxygen-regulated activation step. Abrogation of the Drosophila Egl-9 prolyl hydroxylase homologue, CG1114, caused both stabilization and nuclear localization of Sima, indicating a central involvement in both processes. Tight conservation of the HIF/prolyl hydroxylase system in Drosophila provides a new focus for understanding oxygen homeostasis in intact multicellular organisms.

Active Transport, Cell Nucleus↗
PubMed2002

From erythropoietin to oxygen: hypoxia-inducible factor hydroxylases and the hypoxia signal pathway.

The regulation of blood red cell production by the hormone erythropoietin (Epo) provides a paradigm for control of gene expression by oxygen. Analysis of this pathway has revealed a widespread system of gene regulation based on a transcriptional complex termed hypoxia-inducible factor (HIF). Hydroxylation of specific prolyl and asparinyl residues in the alpha subunit of HIF by a series of non-haem iron-dependent dioxygenases has been defined as a novel mechanism of protein modification that transduces the oxygen-sensitive signal.

Animals↗
PubMedDate not supplied

HIF hydroxylation and cellular oxygen sensing.

Hypoxia-inducible factor (HIF) is a transcriptional complex that mediates a broad range of cellular and systemic responses to hypoxia. Analysis of HIF-alpha subunits has demonstrated that its activity is regulated by a series of oxygen-dependent enzymatic hydroxylations at specific prolyl and asparaginyl residues. Combined structural/genetic approaches have identified the relevant enzymes as members of the 2-oxoglutarate-dependent dioxygenase superfamily, possessing a beta-barrel 'jelly-roll' conformation that aligns a 2-histidine/1-carboxylate iron co-ordination motif at the catalytic centre. HIF prolyl hydroxylation is performed by a closely related set of isoenzymes (PHD1-3) that differ in abundance and subcellular localisation. Hydroxylation of either human HIF-1alpha Pro402 or Pro564 promotes interaction with the von Hippel-Lindau tumour suppressor protein (pVHL). In oxygenated cells this process targets HIF-alpha for rapid proteasomal destruction. HIF asparaginyl hydroxylation is performed by a protein termed factor inhibiting HIF (FIH). In oxygenated cells hydroxylation of human HIF-1alpha Asn803 prevents interaction with the p300 transcriptional co-activator, providing a second mechanism by which HIF-mediated transcription is inactivated. Genetic studies demonstrate a critical function for both types of enzyme in regulating the HIF transcriptional cascade. Limitation of activity in hypoxia supports a central role of these hydroxylases in cellular oxygen sensing. Regulation of the amount of hydroxylase protein, and the supply of other co-substrates and co-factors, particularly the cellular availability of iron, also contribute to tuning the physiological response to hypoxia.

Animals↗
PubMedDate not supplied

Understanding hypoxia signalling in cells--a new therapeutic opportunity?

The possibility that cells possess specific interfaces with molecular oxygen that have a prime function in biological control has long interested biologists. Specific 'oxygen-sensing' mechanisms have been defined in bacteria and yeast, but, until recently, have remained elusive in higher organisms. Studies of hypoxia pathways have now, however, revealed the existence of a series of non-haem Fe(ll) and 2-oxoglutarate-dependent dioxygenases that catalyse oxygen-regulated hydroxylation of specific amino acids in a key transcription factor termed hypoxia-inducible factors (HIFs). These post-translational hydroxylations govern both the proteolytic stability and activity of HIF and therefore the transcription of many hundreds of human genes whose expression changes in accordance with cellular oxygen availability. This paper will review these developments and consider the biological and potential therapeutic implications.

Cell Hypoxia↗