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Symposium Reports |
1 Department of Pediatrics, Division of Neonatology, North-western University, Chicago, IL 60611, USA
Abstract
All eukaryotic cells utilize oxidative phosphorylation to maintain their high-energy phosphate stores. Mitochondrial oxygen consumption is required for ATP generation, and cell survival is threatened when cells are deprived of O2. Consequently, all cells have the ability to sense O2, and to activate adaptive processes that will enhance the likelihood of survival in anticipation that oxygen availability might become limiting. Mitochondria have long been considered a likely site of oxygen sensing, and we propose that the electron transport chain acts as an O2 sensor by releasing reactive oxygen species (ROS) in response to hypoxia. The ROS released during hypoxia act as signalling agents that trigger diverse functional responses, including activation of gene expression through the stabilization of the transcription factor hypoxia-inducible factor (HIF)-
. The primary site of ROS production during hypoxia appears to be complex III. The paradoxical increase in ROS production during hypoxia may be explained by an effect of O2 within the mitochondrial inner membrane on: (a) the lifetime of the ubisemiquinone radical in complex III; (b) the relative release of mitochondrial ROS towards the matrix compartment versus the intermembrane space; or (c) the ability of O2 to access the ubisemiquinone radical in complex III. In summary, the process of oxygen sensing is of fundamental importance in biology. An ability to control the oxygen sensing mechanism in cells, potentially using small molecules that do not disrupt oxygen consumption, would open valuable therapeutic avenues that could have a profound impact on a diverse range of diseases.
(Received 28 June 2006;
accepted after revision 17 July 2006; first published online 20 July 2006)
Corresponding author P. T. Schumacker: Department of Pediatrics, 303 East Chicago Ave, Ward Building 12-191, Chicago, IL 60611, USA. Email: p-schumacker{at}northwestern.edu
The cells of the body require energy for self-repair, upkeep and maintenance, and for sustaining their tissue- or organ-specific functions. The majority of these processes are endothermic and are therefore coupled to the hydrolysis of ATP, which provides the necessary free energy. The cellular pool of ATP and other high-energy phosphates provides a renewable energy supply that is required for survival. The majority of cells maintain the supply of ATP through oxidative phosphorylation, which couples the oxidation of metabolic substrates to the synthesis of ATP from ADP and inorganic phosphate in the mitochondria. That process consumes oxygen in proportion to the rate of ATP utilization by the cells. Hence, increases in metabolic activity are associated with increases in the rate of oxygen utilization. Multicellular organisms have developed elaborate systems for supplying O2 to each cell in accordance with its metabolic activity and for adjusting the delivery when the metabolic needs change, as occurs in exercise. Although cells have a limited ability to generate ATP in the absence of oxygen, the loss of O2 supply, even briefly, threatens survival. Not surprisingly, organisms have evolved a complex array of mechanisms to sustain the supply of oxygen to the cells of the body and to protect themselves in the event that oxygen availability becomes limiting. The ability to sense the level of O2, and to sound the alarm when it falls, is therefore a fundamental requirement for survival of multicellular organisms.
Given the potentially lethal consequences of oxygen deprivation, organisms have developed a number of responses to defend the oxygen supply in response to changes in the environment, or in response to diseases that undermine the transport of O2 to tissues. Well-known examples of specialized oxygen sensing systems include the arterial chemoreceptors that monitor the level of blood oxygenation and that signal the respiratory system to increase the level of alveolar ventilation when arterial partial pressure of O2 (PO2) decreases. Similarly, cells of the liver and kidney adjust the secretion of the hormone erythropoietin in order to regulate the number of circulating erythrocytes to assure adequate tissue oxygenation. Some cells even have the ability to downregulate their oxygen utilization during hypoxia, allowing them to survive in the face of a more severe limitation in O2 supply (Land et al. 1993; Chandel et al. 1997; Budinger et al. 1998). These and other specialized responses support the basal supply of oxygen to tissues, and allow the organism to respond to changes in the environment or in the levels of demand. However, all cells of the body have the ability to detect a decrease in oxygen tension and to initiate gene transcription by activating the hypoxia-inducible factors (Maxwell et al. 1993; Wang & Semenza, 1993).
Although the ventilatory and cardiovascular systems respond immediately to hypoxia or to a change in O2 supply or demand, many of the responses to hypoxia require hours, days or even weeks to be effected. For example, regional hypoxia within muscle triggers the secretion of vascular growth factors, which mediate the growth of new capillaries into the region over a period of days. Decreases in arterial blood oxygen levels activate the transcription of erythropoietin, which stimulates the synthesis and maturation of new erythrocytes over a period of a week or two (Goldwasser et al. 1989). Cellular hypoxia leads to the upregulation of genes involved in glucose uptake, glucose metabolism, cell survival, vascular tone, cytoskeletal organization, apoptosis, extracellular matrix metabolism, cell adhesion and iron metabolism (Wykoff et al. 2000; Hu et al. 2003; Wykoff et al. 2004; Gunton et al. 2005; Greijer et al. 2005). These responses require activation of transcription and subsequent translation of genes, which requires time. Finally, the end-point effects of some hypoxia-induced gene expression, such as angiogenesis, vascular remodelling, cell migration and proliferation, require hours or days to reach completion. Physiologically important responses that cannot be activated within seconds must therefore be initiated early, in anticipation of a condition that could threaten survival if it were to progress. At least some of the responses described above are aimed at preventing or lessening the severity of hypoxia. Thus, to confer protection, these responses must be regulated by a control system that is initiated at the earliest stages of hypoxia. Delaying activation of these systems until cell metabolism becomes limited by oxygen availability is like delaying the activation of a fire alarm until a building is fully engulfed in flames. Hence, a functional requirement for a cellular oxygen sensor is that it must be capable of detecting subtle changes in the level of oxygenation and of initiating a proportional activation of the cellular response.
Transcriptional responses to hypoxia
The principal regulator of transcriptional responses to hypoxia is the hypoxia-inducible factor (HIF) family of transcription factors (Semenza et al. 1991, 1994; Semenza & Wang, 1992). Hypoxia-inducible factor is a heterodimeric complex comprised of an oxygen-regulated subunit (HIF-) and a constitutively expressed ß subunit, also referred to as the arylhydrocarbon receptor nuclear translocator (ARNT; Semenza, 2000). Hypoxia-inducible factor- is constitutively transcribed, and the protein is continuously translated. However, under normoxia it is rapidly degraded by the ubiquitin–proteasome system (Huang et al. 1996, 1998; Salceda & Caro, 1997). Attempts to measure HIF-1 by immunoblot assays of whole-cell protein lysates from normoxic cells reveal HIF-ß protein, but HIF- protein levels are less evident because the subunit is continuously degraded. Under sustained hypoxia, degradation of the HIF- subunit is inhibited, allowing the protein to accumulate, heterodimerize and translocate to the nucleus. There, it binds to specific (hypoxia response element, HRE) consensus sequences in DNA as it assembles in association with other proteins comprising the transactivation complex (Ebert & Bunn, 1998; Semenza, 1999). Reoxygenation of hypoxic cells is associated with a rapid disappearance of the HIF- protein from the cell, subsequent to its ubiquitin labelling and proteasome degradation (Huang et al. 1998). Two oxygen-regulated members of the HIF family include HIF-1 and HIF-2, which are similarly regulated by oxygen (Talks et al. 2000; Sowter et al. 2003; Hu et al. 2003). While HIF-1 and HIF-2 exhibit overlap in their ability to regulate a large number of genes, recent work reveals that their functions are not identical, and that a number of important differences exist in their targets (Hu et al. 2003; Park et al. 2003; Wang et al. 2005; Aprelikova et al. 2006).
Virtually every cell in the body is capable of sensing oxygen and regulating HIF-mediated transcription in response to O2 (Maxwell et al. 1993; Wang & Semenza, 1993). This point is illustrated if one transfects any cell in the body with a reporter gene construct, driven by an HRE in its promoter region. If the cell is subsequently challenged with hypoxia, expression of the reporter gene will be detected. Hypoxia-inducible factor is required for embryonic development, and embryos with homozygous knockout of HIF-1 (Iyer et al. 1998; Ryan et al. 1998) or ARNT (Maltepe et al. 1997) die at mid-gestation.
Hypoxia-inducible factor was discovered by Semenza and colleagues, and identified as an oxygen-dependent transcription factor regulated post-transcriptionally at the level of protein stability (Semenza et al. 1991; Wang & Semenza, 1995). Moreover, a specific region of the HIF- subunit was shown to be required for its responsiveness to hypoxia (Huang et al. 1996; Pugh et al. 1997). Deletion of the oxygen-dependent degradation (ODD) domain from a reporter construct abolished its sensitivity to hypoxia, indicating that the ODD was involved in its regulation by O2. A subsequent discovery by Peter Ratcliffe's laboratory at Oxford identified the von Hippel-Lindau (vHL) protein as the E3 recognition component of the ubiquitin ligase responsible for ubiquitin labelling prior to its degradation (Maxwell et al. 1999; Cockman et al. 2000). Von Hippel-Lindau disease manifests by the development of haemangioblastomas and renal clear cell carcinomas that are highly vascular and exhibit high levels of HIF activation (Mole et al. 2001). This phenotype can be explained by the genetic loss of vHL protein, which is required for the normal degradation of HIF. However, the mechanism responsible for regulating the interaction between HIF and vHL protein in accordance with oxygen still was not known at that point. The search for the mechanism of protein regulation culminated in the discovery that the interaction between HIF- and vHL protein was controlled by the hydroxylation of highly conserved proline residues within the ODD (Jaakkola et al. 2001; Ivan et al. 2001). Hydroxylation of these residues by a family of prolyl hydroxylases facilitates interaction with the ubiquitin ligase and therefore controls protein stability. Consequently, regulation of prolyl hydroxylase activity by oxygen is responsible for controlling the activation of HIF.
Regulation of HIF by prolyl hydroxylases
Prolyl hydroxylases (PH) are a family of mixed function oxidases involved in the post-translational modification that signals HIF- for degradation (Tanimoto et al. 2000; Clifton et al. 2001; Epstein et al. 2001; Bruick & McKnight, 2001; Berra et al. 2003; Appelhoff et al. 2004). These enzymes require 2-oxoglutarate (-ketoglutarate) and O2 as substrates, and they require non-haem iron as a cofactor. Chemical inhibitors that block the activity of PH, such as iron chelators that deprive the enzyme of Fe2+, or dimethyloxallyl glycine (DMOG) that competes with 2-oxoglutarate for binding at the hydroxylase, prevent HIF proline hydroxylation and cause accumulation of HIF- and activation of HIF-dependent gene expression (Ivan et al. 2002). The fact that PH utilizes O2 as a substrate raises the question of whether PH functions as the O2 sensor regulating HIF activation (Kaelin, 2003, 2005; Schumacker, 2005). In vitro studies assessing the oxygen dependence of PH initially revealed a Km for O2 near 250 µM, close to the level of oxygen expected in a solution equilibrated with room air (Hirsilae et al. 2003). If this were identical with the Km
in vivo, one would predict that PH would exhibit a progressive decrease in activity throughout the physiological range of O2 levels. However, studies of the O2 dependence of HIF stabilization show that it begins to be activated at oxygen levels below 5%, with maximal activation near 0.5% O2 (Jiang et al. 1996). One explanation for these discrepancies is that PH in the intact cell may be regulated differently from PH in isolation. Moreover, these in vitro studies utilized a small peptide derived from HIF- as a substrate rather than the full-length protein, which may have introduced important differences in the activity of the enzyme (Hirsilae et al. 2003). A fuller understanding of how PH activity is regulated requires the ability to monitor PH activity (i.e. ODD proline hydroxylation) in the intact cell. This is technically challenging, since the levels of HIF in the cell are low, even under hypoxic conditions. Overexpression of an ODD-containing protein can be employed to remedy that situation, but excessive levels of ODD protein can potentially disrupt normal regulation by overpowering the degradation systems at the PH or vHL protein steps. Work presently underway will undoubtedly address this problem in the future.
Mitochondrial oxygen sensing
While it is clear that PH cannot function without oxygen, an alternative possibility is that another oxygen sensor within the cell is responsible for regulating PH activity during physiological hypoxia. We proposed that mitochondria function as O2 sensors that regulate a wide range of cellular responses to hypoxia, ranging from transcription factor activation to the control of vascular tone in the pulmonary circulation (Chandel et al. 1998). The proposed relationship between mitochondria and PH activity is depicted in Fig. 1. Mitochondria have long been suspected to be the site of oxygen sensing, since they bind O2 at cytochrome oxidase and they represent the primary site of oxygen consumption in the cell (Bunn & Poyton, 1996). However, a conceptual hurdle has been that cytochrome oxidase activity, as indicated by the rate of oxygen consumption by isolated mitochondria, does not become limited by O2 availability until the oxygen concentration falls to about 1 µM (<< 0.1% O2). This behaviour would appear to make the electron transport chain suitable as a detector of anoxia, but unsuitable as a sensor of moderate hypoxia. Clearly, a sensor of anoxia would be incapable of triggering the anticipatory responses described above.
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Subsequently, workers in our laboratory demonstrated that isolated cytochrome oxidase responds to hypoxia by decreasing its maximal turnover rate (Chandel et al. 1996, 1997). This alters the redox state of more proximal complexes in the electron transport chain, potentially allowing the oxidase to function as an oxygen sensor, and to signal by generating reactive oxygen species. This change in cytochrome oxidase activity during hypoxia was linked to a downregulation of metabolic activity in a variety of different cell types (Chandel et al. 1995; Budinger et al. 1998). The decrease in respiration during prolonged hypoxia, a response termed ‘hypoxic conformance of metabolism’, was found to be the result of a decrease in ATP utilization by the cells rather than a limitation in the mitochondrial ability to generate ATP. Based on those findings, we hypothesized that mitochondria generate a signal during hypoxia that elicits a decrease in metabolic activity by the cell. This suppression of metabolic activity leads to a decrease in ATP hydrolysis, leading to a decrease in mitochondrial respiration as a consequence of the lesser return of ADP to the mitochondria.
If cytochrome oxidase were solely responsible response for detecting hypoxia, one would predict that mitochondrial inhibitors that block activity of cytochrome oxidase, or that prevent electron transport from cytochrome c to the oxidase, would abolish the oxygen sensing function. Yet antimycin A, which prevents electron transport from complex III to cytochrome c, and cyanide, which blocks complex IV, do not inhibit the HIF-1 response to hypoxia (Gleadle et al. 1995b; Chandel et al. 1998). Stated differently, in the presence of antimycin A, the more distal complexes remain in a fully oxidized state yet the cell can still respond to hypoxia. Those observations suggested that changes in the redox state of cytochrome oxidase (induced by hypoxia) were not the primary mechanism regulating HIF activation. Our findings revealed that functionality within the middle but not the distal region of the electron transport chain is required for oxygen sensing, although they did not rule out the possibility that redox changes at cytochrome oxidase could contribute secondarily.
Further studies investigating the involvement of the electron transport chain in oxygen sensing revealed that a functional electron transport chain was required for HIF stabilization in hypoxia. This was based on the observation that electron transport inhibitors, including rotenone (an inhibitor of the distal end of complex I), myxothiazol and stigmatellin (inhibitors of the upstream end of complex III), were capable of blocking the stabilization of HIF-1 during hypoxia (Chandel et al. 1998; Agani et al. 2000; Guzy et al. 2005), as were genetic deletions in complex I (DeHaan et al. 2004). Although the pharmacological agents blocked the stabilization of HIF-1 during hypoxia, they did not prevent HIF-1 stabilization in response to the iron chelator deferroximine (DFO). In addition, inhibitors acting at complex IV did not appear to inhibit the response to hypoxia (Gleadle et al. 1995a,b). To avoid the use of pharmacological agents that might have non-specific effects on the cell, we utilized cells that had been depleted of their mitochondrial DNA (
0 cells; Chandel et al. 1998; Chandel & Schumacker, 1999; Guzy et al. 2005; Mansfield et al. 2005). The mitochondrial DNA encodes specific subunit components of complexes I, III, IV and V that are required for electron transport and ATP production. Cells lacking mitochondrial DNA do not exhibit oxidative phosphorylation and they do not respire because the electron transport chain is dysfunctional (Chandel & Schumacker, 1999). Hence, they are forced to supply all of their ATP needs through anaerobic glycolysis. These cells failed to stabilize HIF-1 during hypoxia, yet they still were able to respond to hypoxia mimetics, such as DFO. Those findings were consistent with the notion that at least some functionality in the electron transport chain was required for activating HIF during hypoxia.
Two reports challenged those findings. In one study, Vaux and co-workers incubated 0 cells in hypoxic conditions and found that they were still capable of stabilizing HIF-1 (Vaux et al. 2001). That report suggested that the mitochondrial electron transport was not required for oxygen sensing, and that results obtained using mitochondrial inhibitors may have been due to non-specific toxic effects. However, careful analysis of that paper reveals that they utilized virtual anoxia (0% O2) to test the hypoxic responsiveness, but did not include studies of physiological hypoxia (1–5% O2). Stabilization of HIF in hypoxia normally begins at 
5% O2 and increases exponentially as the O2 concentration falls towards zero (Jiang et al. 1996). Based on work done after publication of the paper by Vaux et al. (2001), we now know that prolyl hydroxylase requires oxygen as a substrate for hydroxylation of HIF-1 (Ivan et al. 2001; Jaakkola et al. 2001). Under anoxic conditions, stabilization of HIF will occur even if the mitochondrial oxygen sensor is disabled by pharmacological or genetic means (Schroedl et al. 2002). Armed with the present understanding of the role of prolyl hydroxylase in the regulation of HIF, we now know that 0 cells will stabilize HIF-1 during anoxia, but fail to activate HIF during physiological hypoxia. In a second study, challenging our report (Chandel et al. 1998), Srinivas and co-workers obtained 0 cells that had been characterized previously by King & Attardi (1989) and examined their response to graded levels of hypoxia in the physiological range (Srinivas et al. 2001). Surprisingly, those cells were capable of activating HIF under moderate hypoxia. However, a later analysis of the same cell line revealed significant contamination with mycoplasma, and elimination of the microbial contamination abolished its sensitivity to physiological hypoxia (personal communication, M. Celeste Simon, University of Pennsylvania). It therefore appears that the contamination of a 0 cell line with micro-organisms can potentially restore responsiveness to hypoxia. In subsequent studies, we have further confirmed that 0 cells that are free from microbial contamination fail to respond to hypoxia, but are still capable of responding to anoxia (Guzy et al. 2005; Mansfield et al. 2005; Brunelle et al. 2005).
Studies using pharmacological inhibitors suffer from the problem that it is difficult to rule out the possibility that non-specific effects may contribute to the response, or that unknown effects of the drug may alter the response. The 0 cells are somewhat more specific, yet the generation of these cells still requires long-term incubation in ethidium bromide, a potential mutagen. To address these weaknesses it is necessary to develop alternative genetic models to test the hypothesis.
In eukaryotes, complex III of the electron transport chain consists of an assembly of 11 proteins encoded by nuclear and mitochondrial genes (Iwata et al. 1998). The function of complex III is to accept electrons from ubiquinol, to transfer these to cytochrome c, and to translocate protons across the inner mitochondrial membrane (Fig. 2). A central component of complex III involves the Q (ubiquinone) cycle. At complex I and II, a pair of reducing equivalents is transferred to ubiquinone, yielding ubiquinol. However, cytochrome c and cytochrome oxidase accept single electrons sequentially. Within complex III, the Q cycle performs the important role of converting the paired transfers of complex I and II into sequential transfers needed for complex IV (Saraste, 1999). A ubiquinol molecule binds to complex III at the Qo site, so-named for its location near the outer surface of the inner membrane. A single electron is transferred to the Rieske iron–sulphur protein, yielding the univalently reduced ubisemiquinone at that site. The iron–sulphur group of the Rieske protein is situated on a hinged region that rotates, allowing the electron to be transferred to the cytochrome c1 centre. Meanwhile, the ubisemiquinone transfers its electron to the b cytochrome, allowing a new ubiquinol molecule to bind. A second quinone reduction site on the inner surface of the membrane (Qi) allows electrons from the b cytochrome to be transferred, regenerating ubiquinol. Thus, in a full cycle, two quinol molecules are consumed at the Qo site, two electrons are transferred sequentially to cytochrome c, and a pair of electrons is transferred to generate ubiquinol at the Qi site. This process is coupled to the transfer of four protons from the matrix to the intermembrane space, contributing to the transmembrane electrochemical gradient (Yu et al. 1998; Trumpower, 2002; Hunte et al. 2003).
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The electrical potential across the inner mitochondrial membrane normally ranges from 180 to 200 mV, with the matrix negative relative to the intermembrane space (Wallace, 2001). It is therefore likely that superoxide anion released from the Qo site would migrate to the intermembrane space. Superoxide generated at the Qi site would be likely to contribute to oxidant stress in the matrix compartment. Superoxide dismutase in the matrix (Mn-SOD) or the intermembrane space (Cu,Zn-SOD) would dismute superoxide to hydrogen peroxide, which has the capacity to cross membranes.
A measure of intracellular ROS production can be obtained by superfusing cells on an inverted fluorescence microscope with media containing the probe dichlorofluorescein diacetate at low concentration, typically 5 µM. Basal oxidation of the probe by various processes in the cell yields the fluorescent product dichlorofluorescin (DCF), which accumulates and can be observed in fluorescence images. The overall cellular intracellular fluorescence in that case represents a balance between the rate of oxidation of the probe (tending to increase fluorescence intensity) and the rate of leakage of oxidized probe from the cell (which decreases fluorescence as it is carried away by the continuous superfusion). Under control conditions, a steady state can be reached wherein the concentration of oxidized probe, and thus the intracellular fluorescence intensity, remains stable over time. If the gas bubbling the media is then switched to a hypoxic mixture, a progressive increase in the intracellular fluorescence is observed over the following 2–20 min (Chandel et al. 1998; Vanden Hoek et al. 1998; Waypa et al. 2002). This suggests that hypoxia causes an increase in the rate of oxidation in the cell. If the oxygen concentration is subsequently returned to normoxia, the intracellular fluorescence decreases, which can be explained by a decrease in the rate of oxidation and a progressive leakage of the oxidized fluorescent dye from the cell. In cardiomyocytes studied in this manner, Duranteau d co-workers observed that the extent of increase in fluorescence was proportional to the severity of hypoxia, such that greater increases were seen with 1% O2 compared with 3 or 5% (Duranteau et al. 1998). These responses were attenuated by rotenone and thenoyltrifluoroacetone (TTFA), inhibitors that block the generation of ubiquinol at complex I and II, respectively. These findings suggested that hypoxia increases ROS production at complex III in the mitochondria. This increase in ROS production was paradoxical, since the concentration of O2, a substrate for ROS production, is decreased under hypoxia. This finding was unexpected, and it reasonably raised questions about the veracity of the data provided by DCF. For example, could the increase in DCF fluorescence be the result of a decrease in the leakage of oxidized probe from the cells during hypoxia? A major weakness of this probe is that the fluorescence signal depends on the concentration of the dye, which can potentially be influenced by a variety of factors (Hockberger et al. 1999).
Addressing this problem required development of a new class of sensors capable of providing a ratiometric measure of redox status. The importance of ratiometric performance was first illustrated in the field of calcium signalling, where a major advance was achieved with the development of fura-2, an ionized Ca2+ sensor (Grynkiewicz et al. 1985). That probe exhibits ratiometric behaviour, such that its emission (510 nm) during excitation at 340 versus 380 nm changes reciprocally with the ionized calcium concentration. The advantage of a ratiometric sensor is that its measure is independent of the concentration of the probe or the intensity of the excitation. We utilized a protein-based Fluorescence Resonance Energy Transfer (FRET) sensor termed HSP-FRET to assess oxidant stress in cells subjected to hypoxia (Guzy et al. 2005). That sensor consists of cyan- and yellow-fluorescent proteins (CFP, YFP) linked by the regulatory domain from a redox-sensitive bacterial heat shock protein (HSP-33). In bacteria, the chaperone activity of HSP-33 is controlled by redox conditions by a cysteine thiol-containing regulatory domain (Graf et al. 2004; Jakob et al. 1999). Under basal conditions in the cell, the thiol groups remain reduced and chaperone activity is inhibited; oxidation of the cysteine thiols by exogenous oxidant stress causes dithiol formation and functional activation of the protein. FRET sensors produce ratiometric data because changes in the conformation of the hinge region results in a change in Forster energy transfer from CFP to YFP. In this regard, separation of the fluorophores causes an increase in CFP intensity and decrease in YFP intensity, whereas approximation of CFP and YFP causes the opposite response. With HSP-FRET, approximately 4% of the fluorescence of YFP under baseline conditions is attributable to energy transfer from CFP. In cells transfected with HSP-FRET, hypoxia (1.5% O2) caused a progressive unfolding of the protein, which was similar to the response elicited by addition of exogenous H2O2. Under anoxic conditions, this response was abrogated. These findings supported the notion that hypoxia elicits an increase in oxidant signalling in cells (Fig. 3). Moreover, overexpression of catalase, using a recombinant adenovirus, attenuated this response, as did addition of chemical antioxidants (Guzy et al. 2005). These findings provide strong support for the conclusion that physiological hypoxia elicits an increase in oxidant production in a wide range of cell types.
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stabilization. Interestingly, cells with significant knockdown of the Rieske protein showed selective loss in responsiveness to hypoxia, yet these cells were not different from mock knockdown controls in their response to the hypoxia mimetics DFO or cobalt chloride, the prolyl hydroxylase inhibitor dimethyloxallyl glycine (DMOG), or anoxia (Fig. 4). Similar results were reported by Brunelle et al. (2005) using transient knockdown of the Rieske protein. Additionally, the Rieske knockdown cells showed a significant attenuation of the ROS response to hypoxia as assessed with HSP-FRET (Guzy et al. 2005; Fig. 5). Like the control cells, Rieske knockdown cells were still capable of stabilizing HIF-1 in response to anoxia, and in response to exogenous oxidants including H2O2, glucose oxidase, and tert-butyl hydroperoxide. These finding provide strong evidence that a functional complex III, and the ROS signal generated by hypoxia, were required for stabilizing HIF-1 during hypoxia, but were not required for anoxia.
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and HIF-2 during hypoxia. During anoxia, neither mitochondrial electron transport nor ROS are required for HIF activation, since the lack of oxygen inhibits prolyl hydroxylase directly. Unresolved questions for the mitochondrial model
To be sure, additional questions remain unanswered with respect to this model. One important challenge will be to identify the signalling pathway linking the mitochondrial oxidant generation to the regulation of prolyl hydroxylase activity. One possibility is that the ROS act directly at the Fe2+ ion associated with prolyl hydroxylase, rendering the enzyme inactive by preventing its redox cycling (Gerald et al. 2004). Another possibility is that phosphorylation or other post-translational modifications of prolyl hydroxylase mediate changes in its activity.
A second unresolved question relates to the molecular mechanism by which hypoxia causes an increase in ROS production from the electron transport chain. Thermodynamically, it would seem improbable that a decrease in substrate concentration would cause an increase in the rate of a simple first-order reaction. However, other factors may come into play, making the process more complex than it would appear to be. Three potential mechanisms could conceivably contribute to this paradoxical response.
The ‘vectoral transport’ hypothesis is based on the observation that ROS generated by complex III can conceivably be released to either the matrix side of the membrane or towards the intermembrane space (Muller et al. 2004). According to this model, molecular oxygen dissolved in the lipid bilayer might alter the balance affecting ROS release such that relatively more oxidants are released to the intermembrane space direction and relatively less are released to the matrix side. Although the specific mechanism responsible for this shift is not known, an oxygen-dependent change in the direction of ROS release could conceivably explain why oxidant stress increases in the cytosol during hypoxia. Interestingly, such a shift could theoretically account for an increase in cytosolic ROS signalling even if overall ROS production were decreased.
The ‘semiquinone lifetime’ hypothesis proposes that O2 interaction with protein or lipids at complex III could regulate the lifetime of ubisemiquinone at the Qo or Qi sites. If the electron removal from ubisemiquinone by the b cytochromes were slowed during hypoxia, the opportunity for superoxide production could increase significantly even if the concentration of oxygen were less. Any small molecule or drug that alters the kinetics of electron removal from the semiquinone to the b cytochromes can potentially affect superoxide generation, even if the oxygen tension is less.
The ‘oxygen access’ hypothesis suggests that hypoxia might increase the access of O2 to the semiquinone moiety at complex III. For example, if the molecular structure of one or more proteins in complex III were affected by the level of oxygen in the membrane such that the ability of O2 to attack the semiquinone were improved under low-oxygen conditions, this could yield an increase in ROS production despite a decrease in the level of available O2.
A common element in each of these models is the requirement that molecular oxygen, or a change in the concentration of oxygen, affect lipid–protein structure in such a way that the transfer of an electron from ubisemiquinone to O2 is increased despite the lowered concentration of oxygen. For ROS production to increase under hypoxia, either the lifetime of the semiquinone must increase or the relative fraction of ROS released to the intermembrane space versus the matrix must increase. Additional studies are needed to fill these gaps in understanding.
Implications for health and disease
Oxygen sensing is a response that is required for development, and for successful transition from placental to lung gas exchange at the time of birth. Without a functional oxygen sensing system, survival into adulthood would be threatened by an inability to respond to changes in environmental oxygen or to changes in oxygen demand as occur during exercise. Oxygen sensing is also important near the end of life, since tumour growth requires angiogenesis which is triggered by the onset of hypoxia within the tumour. In the future, the discovery of small molecules capable of activating the oxygen sensor potentially will open therapeutic approaches for the treatment of conditions as diverse as obstructive sleep apnoea, high altitude sickness, exercise intolerance and therapeutic revascularization. Novel compounds that selectively inhibit the oxygen sensor without blocking mitochondrial respiration have the potential to block not only tumour angiogenesis, but also the other tumour-facilitating genes regulated by HIF-1 and HIF-2 that promote cell survival, metastasis, glycolysis, cell migration, cell proliferation, extracellular matrix metabolism and cell apoptosis. The wide range of processes that is regulated by the oxygen sensing mechanisms of the cell highlights the fundamental importance of this process in health and disease.
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Acknowledgements
This work was supported by the following grants from the National Heart, Lung and Blood Institute, U.S.P.H.S.: HL35440, HL32646, and HL079650
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