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Hypoxia, anoxia, and O2 sensing: the search continues

Paul T. Schumacker

Pulmonary and Critical Care, Department of Medicine, The University of Chicago, Chicago, Illinois 60637


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HYPOXIA ELICITS A WIDE RANGE of adaptive responses at the systemic level, at the tissue level, and at the cellular level. Responses at the systemic level such as increased alveolar ventilation promote survival of the organism by maintaining arterial blood hemoglobin saturation and systemic oxygen transport. Within tissues, hypoxia stimulates the production of vascular growth factors including vascular endothelial growth factor (VEGF), which promote capillary growth and sustain local tissue O2 delivery. At the cellular level, some responses help to enhance organismal survival. For example, hypoxia elicits an increase in the expression and secretion of the hormone erythropoietin, which increases systemic oxygen supply by amplifying the rate of erythrocyte formation. Other cellular responses promote cellular survival by enhancing the expression of glycolytic enzymes, cell membrane glucose transporters, and other genes that tend to protect the cell from more severe oxygen deprivation. All of these responses occur because a decrease in O2 has been detected by an oxygen sensor, and a subsequent signaling pathway has become activated.

From a physiological standpoint, an oxygen sensor needs to be capable of detecting a wide range of decreases in O2 tension ranging from mild hypoxia to complete anoxia. An ability to detect moderate hypoxia is important because many adaptive responses are anticipatory to the extent they seek to prevent or delay the onset of more severe hypoxia. An adaptive response that is only triggered after the cell has become anoxic is of little benefit in preventing the onset of that condition in the first place. On the other hand, from a teleological standpoint it is reasonable to expect the O2 sensor should also continue to function even if anoxia is reached, because cell death does not occur immediately in the face of O2 deprivation. From a biophysical perspective, it would be challenging to design a robust O2 sensor that could detect and signal hypoxia ranging in severity from mild to severe and that would continue to function accurately despite physiological variations in pH and temperature. In fact, no oxygen sensor capable of detecting moderate decreases in O2 has yet been definitively identified in mammalian cells. The search for a physiological O2 sensor represents an important and exciting area of research because of its roles in development, in cell survival and in tumor cell biology.

Although the identity of the oxygen sensors has been elusive, much recent progress has been made in understanding the molecular systems activated in response to hypoxia. Most of the genes that are activated during hypoxia are regulated by the transcription factor hypoxia inducible factor-1 (HIF-1) or its close relatives HIF-2 or HIF-3. First identified by Semenza and colleagues (18, 19, 23), this factor is now recognized as a global regulator of O2 homeostasis in a wide range of multicellular organisms (16). In mammals, a large number of target genes for HIF have been identified; the expression of these genes has important consequences for regulation of cellular metabolism, proliferation, survival, cardiovascular function, and iron homeostasis and erythropoiesis. When activated, HIF functions as a heterodimer comprising an alpha -subunit and a beta -subunit that is identical to the aryl hydrocarbon receptor nuclear transporter. Transgenic mice with homologous deletion of either subunit die at midgestation (11, 14), and significant functional abnormalities are even seen in heterozygous adult mice lacking one allele (10, 20). Given the critical importance of this factor during development and its role in both physiological and pathophysiological states, it is not surprising that much attention has been focused on the mechanisms regulating its activation during states of O2 deprivation.

The regulation of HIF-1 has been summarized recently in several excellent reviews (12, 17). Briefly, both HIF-1alpha and HIF-1beta are constitutively expressed under normoxic as well as hypoxic conditions. Although transcriptional expression of HIF-1alpha has been reported to be amplified during hypoxia, the primary mechanism of regulation has been demonstrated to be posttranslational. In this regard, HIF-1alpha is rapidly degraded under normoxic conditions as a result of its ubiquitin labeling and subsequent degradation by the proteasomal system (7a, 15a). This process is inhibited during hypoxia, allowing the protein to accumulate and to heterodimerize with the beta -subunit. Oxygen-dependent degradation (ODD) of HIF-1alpha involves a functional domain of ~200 amino acids, referred to as the ODD region. This region interacts with the von Hippel-Lindau (VHL) tumor suppressor protein, which functions as a critical component of a multiprotein E3 ubiquitin ligase complex (15). Disruption of the VHL gene in patients with von Hippel-Lindau disease results in a constitutive stabilization of HIF-1alpha during normoxia and an associated activation of HIF-1-regulated genes. Interestingly, these patients frequently develop highly vascular tumors as a consequence of dysregulation of vascular growth factors such as VEGF (5).

A critical question concerning HIF-1alpha degradation relates to how this process is inhibited during hypoxia. Three experimental tools have long been employed by investigators seeking to trigger expression of oxygen-regulated genes. These include hypoxia or administration of cobaltous chloride or iron chelators such as desferrioxamine during normoxia. Each is capable of inducing the stabilization of HIF-1alpha levels and of triggering expression of HIF-1-dependent target genes. Two recent studies demonstrated that the interaction between VHL protein and the ODD of HIF-1alpha was regulated by the hydroxylation of a conserved proline residue within the ODD region (8, 9). During normoxia, this proline is hydroxylated by one or more members of the prolyl hydroxylase family in a reaction requiring iron, oxygen, and 2-oxoglutarate. During hypoxia this process becomes inhibited, preventing the interaction of VHL protein with the ODD region and thereby abrogating ubiquitination and degradation. The identification of prolyl hydroxylase in this system therefore represents an important step in understanding the pathways regulating gene transcription during hypoxia. More recently, Lando et al. (13) identified another O2-dependent posttranslational modification of HIF-1alpha involving hydroxylation of a conserved asparagine in the carboxy-terminal transactivation domain. Like the prolyl hydroxylase system, the asparagine hydroxylase(s) mediating this reaction requires iron, oxygen, and 2-oxoglutarate and is inhibited during hypoxia. Hydroxylation of this asparagine during normoxia interferes with the interaction of HIF-1alpha with the p300 coactivator involved in the transcriptional activation. Therefore, two separate hydroxylation switches need to be flipped for HIF-1-mediated gene expression to occur. The involvement of two seemingly distinct hydroxylases in the regulation of HIF-1alpha seems to provide a significant safeguard against inadvertent activation of the hypoxia pathway.

On the basis of the requirement for iron in the function of prolyl and asparagine hydroxylases, it is tempting to speculate that desferrioxamine mimics the effects of hypoxia by chelating the nonheme iron from the catalytic sites in these hydroxylases. Using similar reasoning, one might argue that cobalt ions could inhibit these enzymes by substituting for iron, thereby inactivating enzyme function. Finally, the requirement for O2 in the hydroxylation reactions suggests that hypoxia inhibits hydroxylation by limiting the availability of this molecule as a substrate for the reactions. If true, this would implicate the hydroxylases as functional O2 sensors in the transcriptional response to hypoxia. As tantalizing as this explanation may seem, it is prudent to carefully examine the compatibility of this idea with other data in the field to see whether a consistent picture emerges. Although there is little doubt that these hydroxylases are central participants regulating the stabilization and function of HIF-1alpha , the factors that regulate these hydroxylases are not yet clear. For example, it is conceivable that the activity of these hydroxylases is regulated by some signaling pathway, making them a downstream target of a separate O2 sensor. Such a system would permit a graded inhibition of the enzymes during progressive hypoxia and would also suggest how other triggers of HIF-1 activation such as growth factors could trigger the response during normoxia. Until the apparent Km of the hydroxylases for O2 in the cell can be determined, the question of whether these hydroxylases function as the sensor of hypoxia as opposed to an important downstream effector cannot be known.

Several disparate models have been proposed to explain how molecular oxygen regulates specific processes such as the regulation of HIF-1alpha . At least two models have implicated reactive oxygen species (ROS) in the signal transduction process upstream from HIF-1alpha stabilization. Interestingly, these two models appear diametrically opposed in terms of the hypothesized increases or decreases in ROS production during hypoxia. In the study by Schroedl and colleagues, one of the current articles in focus (Ref. 15b, see p. L922 in this issue), the Chandel group examined HIF-1alpha stabilization at low O2 levels in a variety of cell types. Their studies were motivated by the previous observation that inhibitors such as rotenone, diphenylene iodonium, and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine that block mitochondrial complex I also abolished the HIF-1 response to hypoxia (1, 2, 6). By contrast, inhibitors of more distal regions of the electron transport chain such as cyanide or antimycin A failed to abolish this response and in some cases actually triggered HIF-1alpha stabilization during normoxia (3). The HIF-1alpha response was also abolished by antioxidants and activated during normoxia by low concentrations of exogenous H2O2, leading to the suggestion that mitochondrial ROS production may trigger a signal transduction pathway leading to HIF-1alpha stabilization. This idea was supported by the observation that ROS generation has been found to increase during hypoxia in cells loaded with oxidant-sensitive fluorochromes. Other oxidative changes in DNA also occur during hypoxia, in support of the idea that ROS production increases (7). Finally, rho 0 cells depleted of mitochondrial DNA lost their response to hypoxia, but they still retained the ability to stabilize HIF-1alpha in response to cobalt or desferrioxamine treatment (2, 3). The DNA in mitochondria encodes specific subunits that are required for a functional electron transport chain, so these rho 0 cells cannot respire and are forced to survive purely by anaerobic glycolysis. Interestingly, they also fail to increase ROS generation during hypoxia (4). Collectively, these results tended to implicate the proximal region of the mitochondrial electron transport chain in the O2 sensing pathway.

The idea that O2-dependent HIF-1 regulation was abolished in mitochondria-deficient rho 0 cells was later challenged by other groups (21, 22) that studied a collection of different mutant or rho 0 cells that exhibited various defects in mitochondrial electron transport. In contrast to the results of Chandel et al. (2, 3), they found that rho 0 cells retained the ability to stabilize HIF-1alpha in hypoxia. If true, this would seem to rule out mitochondria as a potential site of O2 sensing. In either case, how can these differences be resolved? The study by Schroedl et al. (15b) makes an important step in resolving this discrepancy. Chandel et al. (2, 3) proposed that mitochondria may function as an upstream regulator of prolyl hydroxylase through the O2-dependent release of ROS under hypoxic conditions (1-3% O2, PO2 = 7-21 mmHg). On the basis of the prolyl/asparagine hydroxylase model, one would predict that complete anoxia (PO2 = 0 mmHg) should inhibit hydroxylation of HIF-1alpha because O2 is required as a substrate for those reactions, regardless of whether mitochondria were involved in O2 sensing. Careful inspection of the papers by Vaux et al. (22) and Srinivas et al. (21) reveals that they measured HIF-1 in rho 0 cells only under near-anoxic conditions (e.g., 0.1% O2). Schroedl et al. (15b) examine this point by comparing HIF-1alpha responses in rho 0 cells under both hypoxic and anoxic conditions. They again report that rho 0 cells selectively lose the ability to respond to hypoxia but retain the ability to stabilize HIF-1alpha in anoxia. These results suggest that prolyl hydroxylases do not serve as the primary O2 sensor regulating HIF-1alpha protein levels under hypoxic conditions but may indeed serve as the primary O2 sensor during anoxia. An interesting corollary is that mitochondria cannot generate ROS during anoxia, due to the lack of O2 as a substrate for electrons. If mitochondrial ROS production is required to trigger HIF-1alpha stabilization during hypoxia, this response would be lost if the cells became anoxic due to the inability to generate ROS. It is interesting to note that during anoxia the hydroxylases also should fail, allowing the transcription of HIF-1-dependent genes to continue.

A larger question in the field of oxygen sensing relates to whether multiple O2 sensors exist in the same cell, whether different mechanisms of O2 sensing occur in different cells, or whether a single O2 sensor regulates the diverse responses to hypoxia among different mammalian cells. In the end, it could turn out that prolyl and asparagine hydroxylases do function both during hypoxia as well as during anoxia in the cell. Such a finding would indicate that multiple O2 sensors must exist, since many responses to hypoxia, including neurotransmitter release in the carotid body and smooth muscle cell contraction in the pulmonary artery, do not require activation of HIF-1. Moreover, other transcription factors including NF-kappa B, activator protein-1, and p53 are activated during hypoxia by a pathway that appears to be distinct from HIF-1 activation and presumably does not involve proline hydroxylation. Although the prospect of identifying a single unifying mechanism of O2 sensing that can explain the diverse responses to hypoxia in multiple cell types is attractive, accumulating evidence tends to point away from this possibility.


    FOOTNOTES

Address for reprint requests and other correspondence: P. T. Schumacker, Dept. of Medicine MC6026, Univ. of Chicago, 5841 So. Maryland Ave., Chicago, IL 60637 (E-mail pschumac{at}medicine.bsd.uchicago.edu).

10.1152/ajplung.00205.2002


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Am J Physiol Lung Cell Mol Physiol 283(5):L918-L921
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