Oxygen-sensing Pathway Research Articles

Genetics and angiogenesis: the example of von Hippel-Lindau disease

Von Hippel-Lindau (VHL) disease is the main cause of inherited kidney cancer and the model of tumoral angiogenesis. This rare syndrome is caused by germline mutations of the VHL tumor-suppressor gene that predispose to the development of a panel of highly vascularized tumors. Main manifestations include CNS and retinal haemangioblastomas, endolymphatic sac tumors, clear-cell renal cell carcinomas (RCC), phaeochromocytomas and pancreatic neuroendocrine tumors. The VHL gene plays a major role in regulation of the oxygen-sensing pathway by targeting the hypoxia-inducible factor HIF for degradation in proteasome. Somatic inactivation of the VHL gene occurs also in most sporadic RCC. Recent progress are pawing the way for the development of antiangiogenic targeted therapies that have already shown promising results in metastatic sporadic RCC.

Cobalt chloride, a hypoxia‐mimicking agent, targets sterol synthesis in the pathogenic fungus Cryptococcus neoformans

We investigated the effects of the hypoxia-mimetic CoCl2 in the pathogenic fungus Cryptococcus neoformans and demonstrated that CoCl2 leads to defects in several enzymatic steps in ergosterol biosynthesis. Sterol defects were amplified in cells lacking components of the Sre1p-mediated oxygen-sensing pathway. Consequently, Sre1p and its binding partner Scp1p were essential for growth in the presence of CoCl2. Interestingly, high copies of a single gene involved in ergosterol biosynthesis, ERG25, rescued this growth defect. We show that the inhibitory effect of CoCl2 on scp1Delta and sre1Delta cells likely resulted from either an accumulation of non-viable methylated sterols or a decrease in the amount of ergosterol. Similar findings were also observed in the ascomycetous yeast, Schizosaccharomyces pombe, suggesting that the effects of CoCl2 on the Sre1p-mediated response are conserved in fungi. In addition, gene expression analysis revealed limited overlap between Sre1p-dependant gene activation in the presence of CoCl2 and low oxygen. The majority of genes similarly affected by both CoCl2 and low oxygen were involved in ergosterol synthesis and in iron/copper transport. This article identifies the Sre1p pathway as a common mechanism by which yeast cells sense and adapt to changes in both CoCl2 concentrations and oxygen levels.

Sre1p, a regulator of oxygen sensing and sterol homeostasis, is required for virulence in Cryptococcus neoformans

Cryptococcus neoformans is an environmental pathogen requiring atmospheric levels of oxygen for optimal growth. Upon inhalation, C. neoformans disseminates to the brain and causes meningoencephalitis, but the mechanisms by which the pathogen adapts to the low-oxygen environment in the brain have not been investigated. We found that SRE1, a homologue of the mammalian sterol regulatory element-binding protein (SREBP), functions in an oxygen-sensing pathway. Low oxygen decreased sterol synthesis in C. neoformans and triggered activation of membrane-bound Sre1p by the cleavage-activating protein, Scp1p. Microarray and Northern blot analysis demonstrated that under low oxygen, Sre1p activates genes required for ergosterol biosynthesis and iron uptake. Consistent with these regulatory functions, sre1Delta cells were hypersensitive to azole drugs and failed to grow under iron-limiting conditions. Importantly, sre1Delta cells failed to produce fulminating brain infection in mice. Our in vitro data support a model in which Sre1p is activated under low oxygen leading to the upregulation of genes required for sterol biosynthesis and growth in a nutrient-limiting environment. Animal studies confirm the importance of SRE1 for C. neoformans to adapt to the host environment and to cause fatal meningoencephalitis, thereby identifying the SREBP pathway as a therapeutic target for cryptococcosis.

Prolyl hydroxylase inhibitor treatment confers whole‐animal hypoxia tolerance

Recently a family of O(2)-dependent prolyl hydroxylase domain-containing enzymes (PHD) has been identified as a cellular oxygen-sensing mechanism. Reduced prolyl hydroxylase activity initiates a signalling cascade that includes the accumulation, as well as the activation, of hypoxia-inducible factor (HIF-1alpha). In turn the transcription factor HIF-1alpha, and other targets of the PHD, elicit a myriad of incompletely understood cellular responses. In these studies we have tested: (1) whether a small-molecule prolyl hydroxylase inhibitor (PHI) can effectively activate the oxygen-sensing pathway when administered systemically to mice, and (2) whether the activation of the PHD signalling pathway at the cellular level results in whole-animal hypoxic tolerance. Mice received daily injections of the PHI, ethyl-3,4 dihydroxybenzoate (EDHB, 100-250 mg kg(-1)) or vehicle. Tissue levels of HIF-1alpha and the serum levels of the HIF-inducible gene, erythropoietin (EPO), were measured to evaluate PHD-pathway activation. To evaluate hypoxic tolerance, the endurance and survival ability of these animals was tested in sublethal (8% O(2)) and lethal hypoxia (5% O(2)) respectively. Systemic treatment of mice with the PHD inhibitor, EDHB, leads to elevated levels of HIF-1alpha in liver and HIF-inducible EPO in serum, indicating activation of the cellular oxygen-sensing pathway. Animals treated with EDHB display significantly increased viability and enhanced exercise performance in hypoxia. These results demonstrate a novel pharmacological strategy to induce hypoxic tolerance and are the first to demonstrate that the activation of the PHD oxygen-sensing pathway at the cellular level is sufficient to produce a hypoxic-tolerant phenotype at the physiological level of the whole animal.

Activation of the PHD oxygen‐sensing pathway induces a low‐respiring, protected mitochondrial phenotype

In these studies, prolyl hydroxylase inhibitors (PHIs) were used to activate the proline hydroxylase-domain containing (PHD) oxygen-sensing pathway in cardiomyocytes. Addition of cyanide and the glycolytic inhibitor 2-deoxy-D-glucose (2-DG) caused, as expected, the collapse of mitochondrial membrane potential (ΔΨmito) in control cells with only partial recovery upon washout. In contrast, ΔΨmito is partially maintained during metabolic inhibition and recovers completely upon washout in PHI-preconditioned cells. Inclusion of rotenone, but not oligomycin, with cyanide and 2-DG was found to collapse ΔΨmito in PHI-pretreated myocytes. Thus, continued complex I activity was implicated in the maintenance of ΔΨmito in PHI-treated myocytes, while a role for the ‘reverse mode’ operation of the F1Fo-ATP synthase was ruled out. Evidence of fumarate to succinate reduction by complex II in the PHI-treated, but not control myocytes, provides an explanation for the apparent finding of ‘anaerobic’ complex I function. Finally we find that PHI-treatment downregulates basal O2-consumption to only ~15% that of controls. Remarkably, addition of DNP, an uncoupler, does not increase O2-consumption rates in PHI-treated myocytes. We conclude that PHD pathway directs mechanisms that allow anaerobic complex I function and that lead to a low-respiring mitochondrial phenotype that is protected against metabolic insult.

Homocysteine Metabolism in Chuvash Polycythemia.

In Chuvash polycythemia, homozygosity for the 598 C->T mutation in the von Hippel-Lindau gene (VHL) leads to upregulation of hypoxia inducible factor-1a (HIF1a), a transcription factor that mediates cellular responses to hypoxia. This defect in the oxygen-sensing pathway causes increased expression of a broad range of hypoxia-regulated genes. Clinically, Chuvash polycythemia (CP) patients display not only erythrocytosis, but also premature mortality related to cerebrovascular and peripheral thrombotic events. As it is not clear that the thrombophilic nature of CP correlates with elevated hematocrit (Gordeuk et al, Blood 103: 3924, 2004), we postulated that homocysteine may be a contributive factor, as preliminary data suggests that CP homozygotes have elevated plasma homocysteine levels (Sergueva, in preparation). Levels of homocysteine depend on its synthesis, involving S-adenosylmethionine, and its metabolism, either via remethylation to methionine, involving methylenetetrahydrofolate reductase (MTHFR), or via degradation by transsulfuration, involving cystathionine beta-synthase (CBS). Severe MTHFR and CBS deficiencies due to rare homozygous mutations lead to extremely high levels of serum homocysteine and are characterized clinically by a high incidence of thromboembolic complications, in addition to a wide range of other clinical symptoms. A recent microarray analysis that looked at the regulation of gene transcription by HIF-1a revealed that CBS and MTHFR gene expressions appear to be down regulated by hypoxia in endothelial cells (Manalo et al, Blood 105: 659, 2005). Downregulation of the genes responsible for homocysteine metabolism may therefore explain the elevated plasma homocysteine concentrations in CP. As hypoxia-regulated genes are often cell-type specific, we studied several types of easily accessible cells and detected expression of CBS and MTHFR in platelets, granulocytes, and EBV-immortalized lymphocytes in normal controls. In order to quantitate this expression, we used real-time RT-PCR and found no quantitative difference between EBV-immortalized lymphocytes in 4 homozygous CP patients, 3 heterozygote CP patients and 1 control. We then examined the peripheral blood from one CP patient and three controls. Although the numbers were small, the CP granulocytes and platelets showed decreased expression of MTHFR compared to controls, with decreased CBS expression seen in the CP granulocytes. These results suggest that the upregulation of HIF1a seen in CP patients might lead to decreased metabolism of homocysteine, which in turn, might contribute to the increased thromboembolic risk seen in CP. As these findings will need to be confirmed with a larger number of patients, we are currently in the process of collecting all accessible CP samples from the U.S. and from Chuvashia and the Italian island of Ischia (Perrotta et al, Blood 107: 514, 2006). Using the peripheral blood cells and in vitro expanded endothelial cells from these patients (Ingram et al, Blood 104:2752, 2004), we hope to analyze the transcripts and enzyme activity of the genes involved in homocysteine synthesis and metabolism and to correlate these findings with CP plasma homocysteine levels.

The prolyl hydroxylase oxygen-sensing pathway is cytoprotective and allows maintenance of mitochondrial membrane potential during metabolic inhibition

The cellular oxygen sensor is a family of oxygen-dependent proline hydroxylase domain (PHD)-containing enzymes, whose reduction of activity initiate a hypoxic signal cascade. In these studies, prolyl hydroxylase inhibitors (PHIs) were used to activate the PHD-signaling pathway in cardiomyocytes. PHI-pretreatment led to the accumulation of glycogen and an increased maintenance of ATP levels in glucose-free medium containing cyanide. The addition of the glycolytic inhibitor 2-deoxy-d-glucose (2-DG) caused a decline of ATP levels that was indistinguishable between control and PHI-treated myocytes. Despite the comparable levels of ATP depletion, PHI-preconditioned myocytes remained significantly protected. As expected, mitochondrial membrane potential (DeltaPsi(mito)) collapses in control myocytes during cyanide and 2-DG treatment and it fails to completely recover upon washout. In contrast, DeltaPsi(mito) is partially maintained during metabolic inhibition and recovers completely on washout in PHI-preconditioned cells. Inclusion of rotenone, but not oligomycin, with cyanide and 2-DG was found to collapse DeltaPsi(mito) in PHI-pretreated myocytes. Thus, continued complex I activity was implicated in the maintenance of DeltaPsi(mito) in PHI-treated myocytes, whereas a role for the "reverse mode" operation of the F(1)F(0)-ATP synthase was ruled out. Further examination of mitochondrial function revealed that PHI treatment downregulated basal oxygen consumption to only approximately 15% that of controls. Oxygen consumption rates, although initially lower in PHI-preconditioned myocytes, recovered completely upon removal of metabolic poisons, while reaching only 22% of preinsult levels in control myocytes. We conclude that PHD oxygen-sensing mechanism directs multiple compensatory changes in the cardiomyocyte, which include a low-respiring mitochondrial phenotype that is remarkably protected against metabolic insult.

Atrazine Stimulates Hemoglobin Accumulation in Daphnia magna: Is it Hormonal or Hypoxic?

Hemoglobin accumulation in daphnids is an important adaptive response that is regulated by at least two distinct molecular pathways: an endocrine pathway stimulated by terpenoid hormones and an oxygen-sensing pathway involving the hypoxia-inducible factor. We found that the herbicide atrazine elevated hemoglobin levels in Daphnia magna and hypothesized that atrazine induced hemoglobin in daphnids through the hormonal regulatory pathway. This hypothesis was tested by modeling the combined effects of atrazine and the terpenoid hormone mimic pyriproxyfen on hemoglobin mRNA levels assuming the same mechanism of action (concentration addition model) and alternatively, assuming different mechanisms of action (response addition model). Model predictions were then compared to experimental assessments of the combined action of these two chemicals on hemoglobin mRNA levels. Changes in hemoglobin expression were evaluated using real-time RT PCR with primers specific to each of three D magna hemoglobin genes (dhb1, dhb2, and dhb3). Both atrazine and pyriproxyfen significantly elevated levels of the hb2 gene product, while having little effect on hb1 and hb3 gene products. Induction of dhb2 by combinations of atrazine and pyriproxyfen did not conform to the concentration addition predictions. Rather, dhb2 induction by these binary combinations was highly consistent with response addition model predictions. These results indicate that atrazine does not induce hemoglobin through the terpenoid hormone-signaling pathway. Results from this study demonstrate that mixtures modeling can be used to assess a chemical's mechanism of action and that atrazine likely stimulates hemoglobin accumulation through the oxygen-sensing pathway.

The VHL/HIF oxygen-sensing pathway and its relevance to kidney disease

Over the past decade major advances have been made in our understanding of the molecular machinery that mammalian cells use to sense and to adapt to a low-oxygen environment. A critical mediator of cellular adaptation to hypoxia is hypoxia-inducible factor (HIF), a basic helix-loop-helix transcription factor that consists of an oxygen-sensitive alpha-subunit, HIF-alpha and a constitutively expressed beta-subunit, HIF-beta. Under conditions of normal oxygen tension, the HIF-alpha subunit is hydroxylated by specific prolyl-hydroxylases and targeted for rapid proteasomal degradation by the von Hippel-Lindau (VHL) tumor suppressor, which is the substrate recognition component of an E3-ubiquitin ligase. In a hypoxic environment or in the absence of functional VHL tumor suppressor protein irrespective of oxygen concentration, HIF-alpha is not degraded and translocates to the nucleus, where it dimerizes with HIF-beta to form transcriptionally active HIF. As a transcription factor, HIF is involved in the regulation of many biological processes that facilitate both oxygen delivery and adaptation to oxygen deprivation by regulating genes that are involved in glucose uptake and energy metabolism, angiogenesis, erythropoiesis, cell proliferation and apoptosis, cell-cell and cell-matrix interactions, and barrier function. This review summarizes some of the most recent advances in the VHL/HIF field and discusses their relevance for pathogenesis and treatment of acute ischemic renal failure, renal fibrosis, and renal cancer.

Sterol Regulatory Element Binding Protein Is a Principal Regulator of Anaerobic Gene Expression in Fission Yeast

Fission yeast sterol regulatory element binding protein (SREBP), called Sre1p, functions in an oxygen-sensing pathway to allow adaptation to fluctuating oxygen concentrations. The Sre1p-Scp1p complex responds to oxygen-dependent sterol synthesis as an indirect measure of oxygen availability. To examine the role of Sre1p in anaerobic gene expression in Schizosaccharomyces pombe, we performed transcriptional profiling experiments after a shift to anaerobic conditions for 1.5 h. Of the 4,940 genes analyzed, expression levels of 521 (10.5%) and 686 (13.9%) genes were significantly increased and decreased, respectively, under anaerobic conditions. Sre1p controlled 68% of genes induced > or = 2-fold. Oxygen-requiring biosynthetic pathways for ergosterol, heme, sphingolipid, and ubiquinone were primary targets of Sre1p. Induction of glycolytic genes and repression of mitochondrial oxidative phosphorylation genes largely did not require Sre1p. Using chromatin immunoprecipitation, we demonstrated that Sre1p acts directly at target gene promoters and stimulates its own transcription under anaerobic conditions. sre1+ promoter analysis identified two DNA elements that are both necessary and sufficient for oxygen-dependent, Sre1p-dependent transcription. Interestingly, these elements are homologous to sterol regulatory elements bound by mammalian SREBP, highlighting the evolutionary conservation between Sre1p and SREBP. We conclude that Sre1p is a principal activator of anaerobic gene expression, upregulating genes required for nonrespiratory oxygen consumption.

Oxygen‐sensing pathway for SK channels in the ovine adrenal medulla

1. The intracellular pathways that modulate the opening of oxygen-sensitive ion channels during periods of hypoxia are poorly understood. Different tissues appear to use either NADPH oxidase or a rotenone-sensitive mechanism as an oxygen sensor. The aim of the present study was to identify the oxygen-sensing pathway in the oxygen-sensitive sheep adrenal medullary chromaffin cell (AMCC). 2. The whole-cell patch-clamp technique was used to measure K+ currents in dissociated adult ovine chromaffin cells as well as SK channel currents expressed in the H4IIE cell line. 3. Diphenyliodonium, an inhibitor of NADPH oxidase, had no effect on the hypoxia-evoked closure of K+ channels in primary AMCC, whereas the mitochondrial inhibitor rotenone abolished the hypoxia-evoked response. Both these compounds significantly reduced K+ current amplitude under normoxic conditions. 4. One possible mechanism through which the oxygen sensor may modulate K+ channel activity is by altering the redox state of the cell. In sheep AMCC, altering the redox state by the addition of H2O2 to the extracellular solution increased K+ conductance. 5. The oxygen-sensitive K+ (Ko2) channels in sheep chromaffin cells are from the SK family and the whole-cell conductance of cells expressing mouse SK2 or SK3, but not human SK1, was increased by H2O2 and decreased by the reducing agent dithiothreitol. 6. These studies show that, in sheep AMCC, Ko2 channels are modulated via a rotenone-sensitive mechanism and that alteration of the cellular redox state mimics the change produced by alterations in Po2. In a heterologous expression system, SK2 and SK3 channels, the channels that initiate hypoxia-evoked changes in AMCC function, are modulated appropriately by changes in cellular redox state.

Molecular Targets from VHL Studies into the Oxygen-Sensing Pathway

Inheritance of a faulty von Hippel-Lindau (VHL) tumor suppressor gene is the cause of VHL disease, a rare multisystemic autosomal dominant disorder characterized by the development of hypervascular tumors in a number of organs, including the retina, brain, spine, pancreas, adrenal gland, and the kidney. Recent discoveries have demonstrated that the VHL gene product pVHL serves as a substrate-recognition component of an E3 ubiquitin ligase complex that targets hypoxia-inducible factor (HIF) transcription factor for polyubiquitination and subsequent degradation. Accordingly, tumor cells devoid of functional pVHL show an inappropriate accumulation of HIF, as well as downstream HIF-target genes, such as vascular endothelial growth factor (VEGF), a potent angiogenic factor. Furthermore, HIF has been found to be elevated in many human cancers further underscoring its common significance in oncogenesis. These and other related recent findings have shed significant insight into the mechanisms governing mammalian cellular oxygen homeostasis and how disruptions in this oxygen-sensing pathway can lead to tumorigenesis. Next generation anti-cancer drugs will undoubtedly emerge from our understanding of the molecular pathways governing normal cellular metabolism, growth and differentiation that have gone awry during neoplastic transformation, and studies in VHL disease will serve as one of the proving grounds for the efficacy of 'designer' anti-cancer drugs tailored against the VHL-HIF pathway.

Maladie de von Hippel-Lindau : progrès génétiques et cliniques récents

Von Hippel-Lindau (VHL) disease is a hereditary cancer syndrome that predisposes to the development of a panel of highly vascularized tumors including CNS and retinal hemangioblastomas, endolymphatic sac tumors, clear-cell renal cell carcinomas (RCC), pheochromocytomas and pancreatic neuroendocrine tumors. CNS hemangioblastomas and RCC are the two main life-threatening manifestations. The disease is caused by germline mutations in the VHL tumor-suppressor gene that plays a major role in regulating the oxygen-sensing pathway by targeting the hypoxia-inducible factor HIF for degradation in proteasome. Somatic inactivation of the VHL gene occurs also in most sporadic RCC and sporadic CNS hemangioblastomas. The demonstration of the critical role of VHL in angiogenesis is paving the way for the development of new specific drugs that could represent an attractive potential treatment for VHL but also for sporadic RCC and other cancers.

Hypoxia-induced neutrophil survival is mediated by HIF-1α–dependent NF-κB activity

Neutrophils are key effector cells of the innate immune response and are required to migrate and function within adverse microenvironmental conditions. These inflammatory sites are characterized by low levels of oxygen and glucose and high levels of reductive metabolites. A major regulator of neutrophil functional longevity is the ability of these cells to undergo apoptosis. We examined the mechanism by which hypoxia causes an inhibition of neutrophil apoptosis in human and murine neutrophils. We show that neutrophils possess the hypoxia-inducible factor (HIF)-1α and factor inhibiting HIF (FIH) hydroxylase oxygen-sensing pathway and using HIF-1α–deficient myeloid cells demonstrate that HIF-1α is directly involved in regulating neutrophil survival in hypoxia. Gene array, TaqMan PCR, Western blotting, and oligonucleotide binding assays identify NF-κB as a novel hypoxia-regulated and HIF-dependent target, with inhibition of NF-κB by gliotoxin or parthenolide resulting in the abrogation of hypoxic survival. In addition, we identify macrophage inflammatory protein-1β as a novel hypoxia-induced neutrophil survival factor.

Role ofVHLGene Mutation in Human Cancer

Germline inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene causes the von Hippel-Lindau hereditary cancer syndrome, and somatic mutations of this gene have been linked to the development of sporadic hemangioblastomas and clear-cell renal carcinomas. The VHL tumor suppressor protein (pVHL), through its oxygen-dependent polyubiquitylation of hypoxia-inducible factor (HIF), plays a central role in the mammalian oxygen-sensing pathway. This interaction between pVHL and HIF is governed by post-translational prolyl hydroxylation of HIF in the presence of oxygen by a conserved family of Egl-nine (EGLN) enzymes. In the absence of pVHL, HIF becomes stabilized and is free to induce the expression of its target genes, many of which are important in regulating angiogenesis, cell growth, or cell survival. Moreover, preliminary data indicate that HIF plays a critical role in pVHL-defective tumor formation, raising the possibility that drugs directed against HIF or its downstream targets (such as vascular endothelial growth factor) might one day play a role in the treatment of hemangioblastoma and renal cell carcinoma. On the other hand, clear genotype-phenotype correlations are emerging in VHL disease and can be rationalized if pVHL has functions separate from its control of HIF.

Catalytic Properties of the Asparaginyl Hydroxylase (FIH) in the Oxygen Sensing Pathway Are Distinct from Those of Its Prolyl 4-Hydroxylases

The activity of hypoxia-inducible transcription factor HIF, an alphabeta heterodimer that has an essential role in adaptation to low oxygen availability, is regulated by two oxygen-dependent hydroxylation events. Hydroxylation of specific proline residues by HIF prolyl 4-hydroxylases targets the HIF-alpha subunit for proteasomal destruction, whereas hydroxylation of an asparagine in the C-terminal transactivation domain prevents its interaction with the transcriptional coactivator p300. The HIF asparaginyl hydroxylase is identical to a previously known factor inhibiting HIF (FIH). We report here that recombinant FIH has unique catalytic and inhibitory properties when compared with those of the HIF prolyl 4-hydroxylases. FIH was found to require particularly long peptide substrates so that omission of only a few residues from the N or C terminus of a 35-residue HIF-1alpha sequence markedly reduced its substrate activity. Hydroxylation of two HIF-2alpha peptides was far less efficient than that of the corresponding HIF-1alpha peptides. The K(m) of FIH for O(2) was about 40% of its atmospheric concentration, being about one-third of those of the HIF prolyl 4-hydroxylases but 2.5 times that of the type I collagen prolyl 4-hydroxylase. Several 2-oxoglutarate analogs were found to inhibit FIH but with distinctly different potencies from the HIF prolyl 4-hydroxylases. For example, the two most potent HIF prolyl 4-hydroxylase inhibitors among the compounds studied were the least effective ones for FIH. It should therefore be possible to develop specific small molecule inhibitors for the two enzyme classes involved in the hypoxia response.

Deciphering the Oxygen Sensing Pathway by Microscopy

This chapter focuses on discovering oxygen sensing pathways by microscopy. Under normoxic conditions, O 2 sensing leads to an optimizing of cell function as exemplified by an enhanced phosphoenol pyruvate carboxykinase (PCK) expression in the periportal liver zone with high PO 2 levels and of enhanced glukokinase (GK) expression in the perivenous liver zone with low PO 2 levels. Under arterial hypoxia tissue, PO 2 frequency distribution is left shifted, leading to a hypoxic response of O 2 sensing for adaptation of cell function. In this chapter, three optical methods used to identify, locate, and image several putative members of the oxygen sensing pathway and their reaction to hypoxia are discussed. The three optical methods are based on light absorption photometry, the optical probe technique, and two-photon confocal laser microscopy. The described microscopic techniques are unique tools to reveal nature, regulation, and localization of critical members of the oxygen sensing pathway. Three-dimensional imaging of intracellular oxygen sensing and concepts related to two photon confocal laser-scanning microscopy and intracellular localization of prolyl hydroxylases are described.

Activation of the Prolyl Hydroxylase Oxygen-sensor Results in Induction of GLUT1, Heme Oxygenase-1, and Nitric-oxide Synthase Proteins and Confers Protection from Metabolic Inhibition to Cardiomyocytes

Recently an oxygen-sensing/transducing mechanism has been identified as a family of O2-dependent prolyl hydroxylase domain-containing enzymes (PHD). In normoxia, PHD hydroxylates a specific proline residue that directs the degradation of constitutively synthesized hypoxia-inducible factor-1alpha. During hypoxia, the cessation of hydroxylation of this proline results in less degradation and thus increases hypoxia-inducible factor-1alpha protein levels. In this study we have examined the consequences of activating the PHD oxygen-sensing pathway in cultured neonatal myocytes using ethyl-3,4 dihydroxybenzoate and dimethyloxalylglycine, inhibitors that, similar to hypoxia, inhibit this family of O2-dependent PHD enzymes. Increased glucose uptake and enhanced glycolytic metabolism are classical cellular responses to hypoxia. Ethyl-3,4 dihydroxybenzoate treatment of cardiomyocyte cultures for 24 h increased [3H]deoxy-4-glucose uptake concurrent with an induction of GLUT1 protein. In addition, ethyl-3,4 dihydroxybenzoate, dimethyloxalylglycine, and hypoxia treatments were found to induce protein levels of nitricoxide synthase-2 and heme oxygenase-1, two important cardioregulatory proteins whose expression in response to hypoxic conditions is poorly understood. In conjunction with these changes in gene expression, activation of the PHD oxygen-sensing mechanism was found to preserve myocyte viability in the face of metabolic inhibition with cyanide and 2-deoxyglucose. These results point to a key role for the PHD pathway in the phenotypic changes that are observed in a hypoxic myocyte and may suggest a strategy to pharmacologically induce protection in heart.

HIF hydroxylation and the mammalian oxygen-sensing pathway

HIF hydroxylation and the mammalian oxygen-sensing pathway

HIF hydroxylation and the mammalian oxygen-sensing pathway.

Hypoxia Hypoxia refers to below-normal levels of oxygen in air, blood, or tissue. Tissue hypoxia leads to cellular dysfunction and ultimately can lead to cell death. Causes of tissue hypoxia include (a) decreased blood oxygenation (such as occurs in certain pulmonary disorders), (b) altered oxygen release from hemoglobin (associated with some hemoglobinopathies), and (c) impaired blood delivery leading to localized anemia (i.e., ischemia) as a result of low cardiac output or vascular obstruction. In order to adapt to hypoxia, mammals use a number of physiological responses. These include (a) increased production of erythropoietin (EPO), which augments the production of red blood cells; (b) induction of tyrosine hydroxylase, which facilitates the control of ventilation through the carotid body; and (c) the stimulation of new blood vessels by upregulation of VEGF (1). At the cellular level, hypoxia induces a number of metabolic changes that allow for continued energy generation despite decreased oxygen availability.