Ebook studies on renal disorders part

Part IV Hypoxia Chapter 21 Involvement of Hypoxia Inducible Factor 1 in Physiological and Pathological Responses to Continuous and Intermittent Hypoxia Role of Reactive Oxygen Species Gregg L Semenza[.]

Part IV Hypoxia

Chapter 21

Involvement of Hypoxia-Inducible Factor 1

in Physiological and Pathological Responses

to Continuous and Intermittent Hypoxia: Role

of Reactive Oxygen Species

Gregg L Semenza

Abstract The hypoxia-inducible factors (HIFs) are transcriptional activators that mediate homeostatic responses to hypoxia At the cellular level, HIF-1 mediates adaptive metabolic responses to hypoxia that serve to maintain energy and redox homeostasis by reducing mitochondrial generation of reactive oxygen species (ROS) At the systemic level, HIFs control erythropoiesis and thereby maintain blood O2-carrying capacity and delivery of O2to body tissues In contrast to these adaptive responses, patients with obstructive sleep apnea are subjected to chronic intermittent hypoxia, a nonphysiological stimulus that induces HIF-1, which med-iates a maladaptive response, systemic hypertension

Keywords HIF-1
Redox
Cytochrome-c oxidase

1 Introduction: Defining Hypoxia

The normal O2concentration to which cells in the human body are exposed varies from ~21% (corresponding to a partial pressure (PO2) of ~150 mmHg at sea level)

in the upper airway to ~1% at the corticomedullary junction of the kidney Biolo-gists usually maintain tissue culture cells in 20% O2(95% air and 5% CO2) and refer to this concentration as normoxia despite the fact that most cells in the human body are exposed to much lower O2levels Whatever the specific set point, complex

Vascular Program, Institute for Cell Engineering;

McKusick Nathans Institute of Genetic Medicine;

and Departments of Pediatrics, Medicine, Oncology, Radiation Oncology,

and Biological Chemistry, The Johns Hopkins University School of Medicine,

Baltimore, MD 21205, USA

e mail: gsemenza@jhmi.edu

Research and Clinical Practice, DOI 10.1007/978 1 60761 857 7 21,

# Springer Science+Business Media, LLC 2011

409

homeostatic mechanisms serve to maintain the cellular O2concentration within a narrow range in vivo

Hypoxia is defined as a reduction in the amount of O2available to a cell, tissue,

or organism As such, it is a relative term Hypoxia can occur continuously (e.g., when individuals ascend to high altitude) or intermittently (e.g., in individuals with sleep apnea, in whom airway obstruction transiently blocks O2uptake, resulting in a rapid decline in blood PO2(hypoxemia), which causes the individual to awaken and resume breathing) Hypoxia can be divided into an acute phase, in which rapid but transient responses are mediated through the posttranslational modification of existing proteins, and a chronic phase, in which delayed but durable changes are mediated through altered gene transcription and protein synthesis Finally, hypoxia can be systemic, as in the case of ascent to high altitude, or local, as in the case of myocardial ischemia associated with coronary artery disease

Ultimately, hypoxia impacts the functioning of individual cells Humans and other metazoan organisms are sustained by energy generated through the oxidative metabolism of glucose and fatty acids in the mitochondria, which results in the production of reducing equivalents that are used to maintain an electrochemical gradient that drives adenosine triphosphate (ATP) synthesis This highly efficient mechanism for producing ATP is dependent upon the utilization of O2 as the terminal electron acceptor at complex IV of the respiratory chain When electrons react with O2prematurely (e.g., at complex III), reactive oxygen species (ROS) are generated Tonic, low-level production of ROS represents a signal that mitochon-drial function is intact, whereas increased ROS production, resulting from reduced

or fluctuating O2availability, is a danger signal that the cell is at risk of oxidative damage and, if uncorrected, of death Thus, many different adaptive responses are triggered by hypoxia, principally through the activity of hypoxia-inducible factor 1 (HIF-1), which is a transcription factor that functions as a master regulator of oxygen homeostasis

2 Molecular Mechanisms of Oxygen Sensing:

The PHD–VHL–HIF-1 Pathway

HIF-1 is a heterodimeric protein that is composed of a constitutively expressed HIF-1b subunit and an O2-regulated HIF-1a subunit [1,2] Under normoxic conditions, the HIF-1a subunit is synthesized and subjected to hydroxylation on proline residue 402

or 564 by prolyl hydroxylase domain (PHD) proteins (principally PHD2) that use O2 and a-ketoglutarate as substrates to catalyze a reaction in which one oxygen atom is inserted into the proline residue and the other oxygen atom is inserted into a-ketoglu-tarate (also known as 2-oxoglua-ketoglu-tarate) to form succinate and CO2[3] Prolyl hydroxyl-ation is required for the binding of the von Hippel Lindau protein (VHL), which recruits a ubiquitin ligase complex [4 7] Ubiquitination marks HIF-1a for degrada-tion by the proteasome [8] FIH-1 binds to HIF-1a and negatively regulates

transactivation function [9] by hydroxylating asparagine residue 803, which blocks the interaction of the HIF-1a transactivation domain with the coactivator p300 or CBP [10] Thus, both the stability and transcriptional activity of HIF-1 are negatively regulated by O2-dependent hydroxylation

When cells are acutely subjected to hypoxia, the hydroxylation reactions are inhibited as a result of substrate (O2) deprivation or increased mitochondrial production of ROS, which may inhibit the hydroxylases by oxidizing a ferrous ion in the catalytic site [3,11] The loss of hydroxylase activity increases HIF-1a stability and transactivation function, leading to its dimerization with HIF-1b, binding of HIF-1 to its recognition sequence 50-(A/G)CGTG-30 [12] in target

genes and increased transcription of target gene sequences into mRNA

Using the HIF-1a DNA sequence to search databases, DNA sequences encoding

a related protein, now designated HIF-2a, were identified [13 16] HIF-2a is also expressed in an O2-regulated manner and dimerizes with HIF-1b [16,17] HIF-1a and HIF-1b are ubiquitously expressed [18], whereas HIF-2a expression is restricted

to a limited number of cell types, including cells of the developing lung, vascular endothelial cells, renal interstitial cells, hepatocytes, cardiomyocytes, and astrocytes [13 16] Whereas HIF-1a homologues are present in all metazoan species studied (including Caenorhabditis elegans, which consists of only ~1,000 cells and contains no specialized systems for oxygen delivery), it appears that HIF-2a arose coincident with the evolution of complex respiratory and circulatory systems in vertebrate organisms

3 Cellular Oxygen Homeostasis: Regulation of Glucose

and Energy Metabolism

Individual cells must adapt to O2deprivation by reprogramming their metabolism The metabolic alterations that are induced by hypoxia are profound Perhaps the most subtle adaptation identified thus far is a subunit switch that occurs in cyto-chrome-c oxidase (COX; complex IV), in which the COX4-1 regulatory subunit is replaced by the COX4-2 isoform as a result of the HIF-1-mediated transcriptional activation of genes encoding COX4-2 and LON, a mitochondrial protease that is required for the hypoxia-induced degradation of COX4-1 [19] This subunit switch serves to optimize the efficiency with which COX transfers electrons to O2under hypoxic conditions Remarkably, the budding yeastSaccharomyces cerevisiae also switches COX subunits in response to hypoxia [20], but does so by a completely different molecular mechanism since yeast do not have a HIF-1 homologue The similar regulation of COX activity in yeast and human cells indicate that the selection for O2-dependent homeostatic regulation of mitochondrial respiration is ancient and likely to be shared by all eukaryotic organisms [19]

A more drastic alteration is the shunting of pyruvate away from the mitochondria

by the HIF-1-mediated activation of the PDK1 gene encoding pyruvate

dehydrogenase (PDH) kinase 1 [21,22], which phosphorylates the catalytic subunit of PDH, the enzyme that converts pyruvate into acetyl coenzyme A (AcCoA) for entry into the mitochondrial tricarboxylic acid cycle, which generates reducing equivalents that are donated to the electron transport chain The reduced delivery of substrate to the mitochondria for oxidative phosphorylation results in reduced ATP synthesis, which must be compensated for by increased glucose uptake via glucose transporters and increased conversion of glucose to lactate by the activity of glycolytic enzymes and lactate dehydrogenase A, which are all encoded by HIF-1 target genes [23 28] Induction ofPDK1 expression will inhibit the oxidative metabolism of AcCoA derived from glucose but will not affect the oxidative metabolism of AcCoA derived from fatty acids The most dramatic response to persistent hypoxia is the active destruction of mitochondria by selective mitochondrial autophagy [29] Remarkably, mouse embryo fibroblasts cultured at 1% O2reduce their mitochon-drial mass by ~75% within 48 h through autophagy that is initiated by the HIF-1-dependent expression of BNIP3, a mitochondrial protein that competes with Beclin1 for binding to Bcl2, thereby freeing Beclin1 to trigger autophagy [29] The adaptive significance of these metabolic responses to hypoxia were revealed

by the finding that HIF-1a-deficient mouse embryo fibroblasts die when cultured under hypoxic conditions for 72 h, due to dramatically increased levels of ROS [21,

28] The cells can be rescued by overexpression of PDK1 or BNIP3, or by treatment with free radical scavengers [21,29] It has long been known that mitochondrial production of ROS increases under hyperoxic conditions [30] However, recent studies have demonstrated that acute hypoxia also leads to increased mitochondrial production of ROS, which is required for the inhibition of HIF-1a hydroxylase activity [11] Exposure of wild-type mouse embryo fibroblasts to hypoxia for 48 h results in reduced levels of ROS, in contrast to HIF-1a-deficient in which the levels

of ROS are markedly increased [21,29]

The following conclusions can be drawn regarding the metabolic adaptation to hypoxia The increase in glycolysis and decrease in respiration that occur in response to hypoxia do not represent a passive effect of substrate (O2) deprivation but instead represent an active response of the cell to counteract the reduced efficiency of respiration under hypoxic conditions, which in the absence of adapta-tion results in the accumulaadapta-tion of toxic levels of ROS These studies indicate that a major role of HIF-1 is to establish, at any O2concentration, the optimal balance between glycolytic and oxidative metabolism that maximizes ATP production without increasing levels of ROS Finally, analysis of lung tissue from nonhypoxic Hif1a+/ mice, which are heterozygous for a HIF-1a null allele and thus partially HIF-1a deficient, revealed a ~50% decrease in mitochondrial mass compared to

WT littermates [28] This remarkable finding indicates that HIF-1 regulates mito-chondrial metabolism even in the tissue exposed to the highest PO2, indicating that HIF-1 performs this critical function over the entire range of physiological PO2 Thus, HIF-1 maintains the metabolic/redox homeostasis that is essential metazoan cells to live with O

4 Systemic Oxygen Homeostasis: Regulation of Erythropoiesis

We discovered HIF-1 in 1992 as a protein required for hypoxia-induced transcrip-tion of the human EPO gene encoding erythropoietin, which is the hormone that controls red blood cell production and thereby determines the O2-carrying capacity

of the blood [31] Red blood cells function to deliver O2from the lungs to every cell

in the body Acute blood loss, ascent to high altitude, and pneumonia each results in

a reduction in the blood O2content The ensuing tissue hypoxia induces HIF-1 activity in cells throughout the body, including specialized cells in the kidney that produce erythropoietin, a glycoprotein hormone that is secreted into the blood and binds to its cognate receptor on erythroid progenitor cells, thereby stimulating their survival and differentiation [32] Analysis of the cis-acting DNA sequences reg-ulating hypoxia-induced EPO gene transcription (the hypoxia response element (HRE)) led to the discovery of HIF-1 as the transacting factor that bound to the HRE [31] Subsequently, HIF-1 has been shown to orchestrate erythropoiesis by coordinately regulating the expression of multiple genes encoding proteins responsible for the intestinal uptake, tissue recycling, and delivery of iron to the bone marrow for its use in the synthesis of hemoglobin, including divalent metal transporter 1 [33], hepcidin [33], ceruloplasmin [34], transferrin [35], and transfer-rin receptor [37,38] Expression of the erythropoietin receptor is also regulated by HIF-1 [39]

Erythropoiesis is impaired in Hif1a / (homozygous HIF-1a-null) embryos, and the erythropoietic defects in HIF-1a-deficient erythroid colonies could not be corrected by cytokines, such as vascular endothelial growth factor or erythropoie-tin, but were ameliorated by administration of iron-salicylaldehyde isonicotinoyl-hydrazone, a compound that can deliver iron into cells independently of iron transport proteins, which was consistent with reduced levels of transferrin receptor

in HIF-1a-deficient embryos and yolk sacs [40] In this study, only yolk sac erythropoiesis could be studied because Hif1a / embryos arrest in their develop-ment on day 8.5 [26] prior to the establishment of definitive erythropoiesis in the liver or bone marrow In contrast, deficiency of HIF-2a (which, like HIF-1a, is O2 -regulated, dimerizes with HIF-1b, and activates target gene expression) has a major effect on EPO production [41] and intestinal iron absorption [33] in adult mice

In humans, familial erythrocytosis is an inherited disorder in which affected individuals produce excess red cells The resulting increased blood viscosity can impair blood flow in cerebral vessels, leading to headache or stroke Four types of familial erythrocytosis have been identified Type 1 is inherited as an autosomal dominant trait and is due to heterozygosity for a mutation in theEPOR gene that results in increased erythropoietin receptor signaling, such as a frameshift that eliminates the last 64 amino acids of the protein [42] Type 2 familial erythrocy-tosis, which is also known as Chuvash polycythemia, is inherited as an autosomal recessive trait and is due to homozygosity for a missense mutation that results in the substitution of tryptophan for arginine at codon 200 of VHL [43] The mutant VHL protein binds to hydroxylated HIF-1a and HIF-2a with reduced affinity, leading to

reduced ubiquitination of HIF-1a and HIF-2a, thereby increasing their steady state levels and the expression of HIF-1 target genes at any given O2concentration Type

3 familial erythrocytosis is an autosomal dominant condition due to heterozygosity for a missense mutation in PHD2 that reduces hydroxylase activity [44] Type 4 familial erythrocytosis is an autosomal dominant condition due to heterozygosity for a missense mutation in HIF-2a that reduces its hydroxylation [45] These findings underscore the critical role of the PHD2 VHL HIF-2a pathway in controlling erythropoiesis in the adult (Fig.1)

5 Pathological Effects of Intermittent Hypoxia

Chronic intermittent hypoxia occurs in individuals with obstructive sleep apnea, in whom airway occlusion results in cessation of breathing leading to hypoxemia, which then arouses the individual to breathe Obstructive sleep apnea may be a contributing factor in 30% of patients with essential hypertension [46] The carotid body is a small chemosensory organ located at the bifurcation of the internal and external carotid arteries that senses arterial PO2 Chronic intermittent hypoxia induces signaling from the carotid body that activates the sympathetic nervous system, leading to increased catecholamine secretion, which increases arterial tone, leading to hypertension [46,47]

HIF-1

Hepcidin Transferrin TransferrinReceptor EPO

Ferroportin

Absorption of iron from intestine

Mobilization of macrophage iron bone marrow cellsIron transport to Erythropoiesis

PHD2 VHL

EPO Receptor

HIF-2

DMT1

Familial Erythrocytosis Type 1 - 2 - 3 - 4

Fig 1 Regulation of erythropoiesis by hypoxia inducible factors (HIF) 1a and 2a HIF 1a and HIF 2a control the expression of multiple genes encoding proteins required for iron absorption and transport and for the survival, proliferation, and differentiation of erythroid cells Molecular

Whereas complete HIF-1a deficiency in Hif1a / ; mice results in embryonic lethality [25, 26], Hif1a+/ heterozygous-null mice develop normally but have impaired responses to hypoxia and ischemia [48 54] Exposure of Hif1a+/ mice and their wild-type littermates to chronic intermittent hypoxia (15 s of hypoxia followed by 5 min of normoxia, 9 episodes per hour, 8 h/day) for 10 days results in marked increases in systolic and diastolic blood pressures and a significant eleva-tion in plasma norepinephrine concentraeleva-tion in the wild-type mice, whereas their Hif1a+/ littermates are unaffected [52] Remarkably, the carotid bodies of Hif1a+/ mice, although structurally and histologically normal, do not respond to hypoxia, although they respond normally to CO2and cyanide [49]

Chronic intermittent hypoxia induces increased production of ROS in rodents [54] and humans [56] and induces HIF-1a expression [52] Administration of the superoxide scavenger manganese tetrakis(1-methyl-4-pyridyl)porphyrin pen-tachloride to wild-type mice reduces the levels of ROS that are generated by chronic intermittent hypoxia [57], blocks the development of hypertension [58], and inhibits the expression of HIF-1a [51] Remarkably, in Hif1a+/ mice, neither HIF-1a expression, ROS generation, nor blood pressure are increased in response to chronic intermittent hypoxia [52] These results indicate that the production of ROS

is required for HIF-1a induction and that HIF-1a induction is required for the production of ROS, suggesting a feed-forward mechanism in which increased levels of ROS induce 1a, which induces more ROS, leading to higher HIF-1a expression

In contrast to the physiological response to continuous hypoxia observed in cultured mouse embryo fibroblasts (described above), in which HIF-1 activity ameliorates increases in ROS levels, the pathological response to chronic intermit-tent hypoxia is characterized by a HIF-1-dependent increase in the levels of ROS Obstructive sleep apnea is a complication of obesity and, like other complications

of obesity, has not been subject to evolutionary selection due to its recent origin Thus, a nonphysiological stimulus (chronic intermittent hypoxia) elicits a maladap-tive response (systemic hypertension) in which HIF-1 contributes to disease patho-genesis

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