Published online before print September 28, 2006, doi: 10.1161/01.RES.0000247068.75808.3f
© 2006 American Heart Association, Inc.
Cellular Biology |
Increases in Mitochondrial Reactive Oxygen Species Trigger Hypoxia-Induced Calcium Responses in Pulmonary Artery Smooth Muscle Cells
From the Department of Pediatrics (G.B.W., R.G., P.T.M., P.T.S.), Division of Neonatology, Northwestern University, Chicago; Department of Pediatrics (J.D.M.), Section of Neonatology, and Department of Medicine (M.W.R., M.M.M.), Pulmonary and Critical Care Section (M.M.M.), The University of Chicago, Ill.
Correspondence to Gregory B. Waypa, Department of Pediatrics, Northwestern University, Ward Bldg 12-189, 303 E Chicago Ave, Chicago, IL 60611. E-mail g-waypa{at}northwestern.edu
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Abstract |
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Mitochondria have been implicated as a potential site of O2 sensing underlying hypoxic pulmonary vasoconstriction (HPV), but 2 disparate models have been proposed to explain their reaction to hypoxia. One model proposes that hypoxia-induced increases in mitochondrial reactive oxygen species (ROS) generation activate HPV through an oxidant-signaling pathway, whereas the other proposes that HPV is a result of decreased oxidant signaling. In an attempt to resolve this debate, we use a novel, ratiometric, redox-sensitive fluorescence resonance energy transfer (HSP-FRET) probe, in concert with measurements of reduced/oxidized glutathione (GSH/GSSG), to assess cytosolic redox responses in cultured pulmonary artery smooth muscle cells (PASMCs). Superfusion of PASMCs with hypoxic media increases the HSP-FRET ratio and decreases GSH/GSSG, indicating an increase in oxidant stress. The antioxidants pyrrolidinedithiocarbamate and N-acetyl-L-cysteine attenuated this response, as well as the hypoxia-induced increases in cytosolic calcium ([Ca2+]i), assessed by the Ca2+-sensitive FRET sensor YC2.3. Adenoviral overexpression of glutathione peroxidase or cytosolic or mitochondrial catalase attenuated the hypoxia-induced increase in ROS signaling and [Ca2+]i. Adenoviral overexpression of cytosolic Cu, Zn-superoxide dismutase (SOD-I) had no effect on the hypoxia-induced increase in ROS signaling and [Ca2+]i, whereas mitochondrial matrix–targeted Mn-SOD (SOD-II) augmented [Ca2+]i. The mitochondrial inhibitor myxothiazol attenuated the hypoxia-induced changes in the ROS signaling and [Ca2+]i, whereas cyanide augmented the increase in [Ca2+]i. Finally, simultaneous measurement of ROS and Ca2+ signaling in the same cell revealed that the initial increase in these 2 signals could not be distinguished temporally. These results demonstrate that hypoxia triggers increases in PASMC [Ca2+]i by augmenting ROS signaling from the mitochondria.
Key Words: hypoxic pulmonary vasoconstriction • reactive oxygen species • redox signaling • antioxidants • fluorescence resonance energy transfer
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Introduction |
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Although hypoxic pulmonary vasoconstriction (HPV) was first described by von Euler and Liljestrand in 1946,1 the underlying mechanism by which vascular cells detect the decrease in O2 tension has not been established. Hypoxia activates an O2 sensor that triggers contraction of pulmonary artery smooth muscle cells (PASMCs) through an increase in cytosolic calcium ([Ca2+]i) via release of Ca2+ from the sarcoplasmic reticulum and/or entry of extracellular Ca2+ through voltage-, receptor-, or store-operated channels in the sarcolemma.2–8 However, the signaling pathways that couple the O2 sensor to the increases in [Ca2+]i have not been established.
Mitochondria have long been considered putative sites of oxygen sensing because they consume O2 and therefore represent the intracellular site with the lowest oxygen tension. Two opposing views have emerged regarding the nature of the O2 sensor and the hypoxia signal transduction pathway. One model is based on measurements of reactive oxygen species (ROS) using lucigenin or luminol chemiluminescence in perfused lungs9 and more recent studies using chemiluminescence, Amplex Red, and 2',7'-dichlorodihydrofluorescein (DCF) in endothelium-denuded rings of distal pulmonary artery (PA).10 Those studies reported finding a decrease in ROS levels during transition from normoxia to hypoxia, leading the authors to hypothesize that decreases in ROS produce a transition in cytosolic redox to a more reduced state.11 This cytosolic reduction has been proposed to close redox-sensitive voltage-dependent potassium (Kv) channels in the plasma membrane, thereby depolarizing the membrane and allowing influx of Ca2+ through voltage-dependent L-type Ca2+ channels. In support of their model, pharmacological reducing agents were found to cause constriction in isolated PA, whereas oxidizing agents produced relaxation.12
An alternate hypothesis is that hypoxia increases ROS generation in PASMCs. This response has been observed in earlier chemiluminescence and DCF fluorescence studies.13–15 Other studies have also detected increases in oxidant production during hypoxia using lucigenin-derived chemiluminescence and electron paramagnetic resonance (EPR) spectrometry.16 The hypoxia-induced increase in oxidants is hypothesized to trigger an increase in [Ca2+]i, resulting in PASMC contraction.17 In support of that model, chemically dissimilar antioxidants or overexpression of catalase block the HPV response,14,16,17 whereas exogenous H2O2 mimics HPV in PASMCs and isolated lungs.14,17
Experimental resolution of the question of whether ROS levels increase or decrease during hypoxia has been problematic because of the technical limitations of the tools used to assess oxidant stress. Fluorescent probes such as DCF and dihydroethidium lack specificity18 and can accumulate within organelles. Similarly, autoxidation and limited intracellular access interfere with the ability of lucigenin or luminol to detect intracellular oxidants.19 Moreover, none of these probes exhibits ratiometric fluorescence, so that a change in intracellular dye concentration or fluorescence path length caused by a change in cell volume will alter fluorescence intensity unrelated to changes in ROS. To address this problem, we assessed cellular redox responses to hypoxia using a ratiometric, redox-sensitive protein sensor, HSP-FRET, expressed in cultured rat PASMCs. This sensor consists of enhanced cyan (CFP) and yellow (YFP) fluorescent protein motifs linked by the redox-dependent regulatory domain from the bacterial heat shock protein HSP-33.20 The HSP-33 domain contains 4 highly conserved cysteine residues coordinating a zinc binding domain. Oxidation of the thiols leads to release of zinc and the formation of 2 disulfides,21 resulting in a structural change in the optical coupling of CFP and YFP. When expressed in cells, this protein provides a sensitive, real-time assessment of changes in redox conditions in the cytosol.22 These studies were complemented with measurements of cellular reduced/oxidized glutathione (GSH/GSSG), which provides an independent assessment of changes in cellular redox balance.
These studies demonstrate that hypoxia triggers an increase in mitochondrial ROS generation from the proximal region of the electron transport chain (ETC). The resulting oxidant stress activates a signal transduction cascade that is necessary and sufficient to cause an increase in cytosolic Ca2+ in PASMCs.
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Materials and Methods |
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An expanded Materials and Methods section is available in the online data supplement at http://circres.ahajournals.org.
Pharmacological Agents and Adenoviruses
Endothelin-1 was obtained from American Peptide; rotenone, myxothiazol, cyanide, ammonium pyrrolidinedithiocarbamate (PDTC), and N-acetyl-L-cysteine (NAC) were obtained from Sigma. Cellular overexpression of enzymatic antioxidants was achieved using recombinant adenoviruses expressing cytosolic catalase and mitochondria catalase,23 as well as Cu, Zn-superoxide dismutase (SOD-I), Mn-SOD (SOD-II), and glutathione peroxidase (GPx1–c-Myc tagged).24–26
Pulmonary Microvessel Myocyte Isolation
PASMCs were isolated from rat lungs as described previously14 using a modification of the method of Marshall et al.13 Cells isolated by this method were confirmed to be PArSMCs as previously described.14 All animals were housed and cared for under National Research Council guidelines for care and use of laboratory animals.
Measurements of GSH and GSSG
GSH and GSSG were measured in PASMCs using a Bioxytech GSH/GSSG-412 kit (Oxis Health Products Inc).
FRET Probes
The HSP-FRET probe was generated by inserting YFP into the pECFP-N1 plasmid (Clontech) between the NheI and BglII sites and then ligating the redox-sensitive regulatory domain from the Escherichia coli HSP-33 between YFP and CFP via the EcoRI and BamHI sites.22 Cells were placed into suspension by trypsinization, then transfected with HSP-FRET using an Amaxa Nucleofector device and plated on glass cover slips. HSP-FRET was excited at 430 nm, whereas fluorescence emission images were obtained at 470 nm (FRET donor, CFP) and 535 (FRET acceptor, YFP) to measure cell redox. Under reducing conditions, the CFP and YFP are in close proximity, and FRET is high. During oxidant stress, oxidation of thiols in the HSP-33 regulatory domain separates the fluorophores and decreases FRET. This increases image intensity at 470 and decreases intensity at 535 nm, resulting in an increase in the 470/535 HSP-FRET ratio.
A genetically encoded FRET-based sensor was used to measure [Ca2+]i. YC2.3 is a high-affinity Ca2+ sensor, consisting of CFP and citrine, a mutant of YFP linked by a calmodulin-M13 hinge region.27–29 When bound to Ca2+, FRET between CFP and citrine increases. An increase in [Ca2+]i is reflected by an increase in the citrine/CFP intensity ratio (535/470). The YC2.3 probe was determined to be unresponsive to exogenously applied oxidants (see the online data supplement). YC2.3 was packaged in a recombinant adenovirus to permit efficient expression of the probe in PASMCs.
Statistics
ANOVA was used to identify significant differences between groups. To control for differences in the hypoxic responses of cultured myocytes, experimental studies and control experiments were always performed on the same day. Statistical significance was set at P<0.05.30
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Results |
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Hypoxia Shifts the Cytosol to a More Oxidized State
To assess the effects of hypoxia on cellular redox status, the ratio of GSH to GSSG was measured in cultured cells. PASMCs were subjected to hypoxia (2 hours at 1.5% O2) in a glove-box, which permitted harvesting of cell lysates without reoxygenation. Hypoxia significantly decreased GSH/GSSG, indicating a shift in the glutathione pool to a more oxidized state (Figure 1). Exogenous H2O2 (20 µmol/L, 15 minutes) also decreased the GSH/GSSG ratio in normoxic cells. The mitochondrial inhibitor myxothiazol prevents electron entry into complex III, thereby preventing ROS generation from complex III or IV. Myxothiazol (100 nmol/L) attenuated the hypoxia-induced decrease in GSH/GSSG without affecting decrease induced by H2O2. These results indicate that hypoxia decreases the ratio of reduced to oxidized glutathione and that this oxidant stress requires electron transport into mitochondrial complex III.
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P<0.05 compared with hypoxic control.
Changes in ROS Signaling and Cytosolic Calcium
PASMCs transfected with the HSP-FRET exhibited a uniform pattern of confocal fluorescence (Figure 2A) that was indistinguishable in appearance from the pattern observed in cells transfected with a nontargeted green fluorescence protein (GFP) expression vector (Clontech EGFP-N1) (see the online data supplement). Although confocal images of PASMCs transduced with YC2.3 exhibited some fluorescence in the nucleus, most of the distribution of the probe was cytosolic (Figure 2E). These images were obtained using excitation laser line at 488 nm and emission at 535 nm. Separate excitation images were obtained for CFP and YFP components of HSP-FRET to confirm colocalization of the fluorophores in PASMCs (see the online data supplement). Individual PASMCs expressing HSP-FRET were imaged every 60 seconds. After establishing a stable baseline under normoxia, the gas bubbling the media was switched to hypoxia (1.5% O2). This elicited an increase in the CFP fluorescence intensity (Figure 2B), a decrease in the YFP fluorescence intensity (Figure 2C), and an increase in the CFP/YFP ratio (Figure 2D) within 3 to 5 minutes, indicating oxidation of the sensor. Individual PASMCs expressing YC2.3 were imaged every 10 seconds. When cells were exposed to hypoxia, citrine fluorescence increased (Figure 2F) and CFP fluorescence decreased (Figure 2G), producing an increase in citrine/CFP ratio (Figure 2H), which was indicative of an increase in [Ca2+]i. Results were summarized by determining the peak increase in HSP-FRET and YC2.3 ratios.
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To determine the contribution of ROS to the HSP-FRET and YC2.3 responses, PASMCs were treated with antioxidants. The hypoxia-induced increase in HSP-FRET ratio was significantly attenuated by PDTC (10 µmol/L) and by NAC (0.5 mmol/L) as was the hypoxia-induced [Ca2+]i increase (Figure 3). PDTC and NAC had no effect on baseline HSP-FRET or YC2.3 ratios during normoxia (see the online data supplement). To assess the role of mitochondrial electron transport, PASMCs were treated with myxothiazol to inhibit complex III or cyanide to inhibit complex IV. Myxothiazol (10 µmol/L) significantly attenuated the hypoxia-induced HSP-FRET and YC2.3 responses, whereas cyanide (1 µmol/L) failed to prevent the increased cytosolic oxidation and augmented [Ca2+]i during hypoxia. Myxothiazol and cyanide had no effect on baseline HSP-FRET or YC2.3 ratios during normoxia (see the online data supplement). Taken together, the results suggest that a cytosolic oxidant signal triggers an increase in [Ca2+]i during hypoxia and that electron flux into complex III is required for the cytosolic oxidant signal, whereas electron transport through complex IV is not necessary.
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Cellular Overexpression of Antioxidant Enzymes Selectively Inhibit HPV
To further assess the role of ROS signaling in HPV, glutathione peroxidase (GPx1) was overexpressed in PASMCs. Western blot analysis for the c-Myc–tagged GPx1 protein revealed dose-dependent expression of GPx1 (Figure 4, inset). GPx1 (5 pfu) significantly decreased the hypoxia-induced increase HSP-FRET oxidation (Figure 4), along with the increase in [Ca2+]i as assessed by YC2.3, although not as effectively as the pharmacological antioxidants.
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To further explore the relationship between hypoxia-induced ROS signaling and the increase in [Ca2+]i, catalase was overexpressed in the cell or the mitochondrial matrix in PASMCs (Figure 5A). Laser-scanning confocal images confirmed the proper targeting of the proteins (Figure 5B through 5D). Both cytosolic and mitochondrial catalase overexpression attenuated the hypoxia-induced increase in both cytosolic oxidant signaling and [Ca2+]i as assessed by the HSP-FRET and YC2.3 responses, respectively (Figure 6). Coexpression of cytosolic and mitochondrial catalase was not more effective than either agent alone (see the online data supplement). This suggests that H2O2 signaling in both cytosol and matrix compartments may contribute to the hypoxia-induced increase in [Ca2+]i. Similarly, SOD-I and SOD-II were overexpressed in PASMCs, and expression levels and targeting were determined by immunoblotting (Figure 5A) and confocal microscopy, respectively (Figure 5E through 5J). Overexpression of SOD-I had no significant effect on either the hypoxia-induced oxidant signaling or [Ca2+]i response to hypoxia, whereas overexpression of SOD-II significantly augmented the [Ca2+]i response but, like SOD I, had no effect on the hypoxia-induced oxidant signaling (Figure 6). These results suggest that the HSP-FRET probe is not responsive to superoxide and that superoxide production leading to H2O2 in the mitochondria contributes to the hypoxia-induced increase in [Ca2+]i.
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Simultaneous Comparison of ROS and Calcium Signaling
Assessment of [Ca2+]i and ROS signals in the same cell using HSP-FRET and YC2.3 is precluded by their common use of CFP and YFP. We therefore used Fura-2 to assess [Ca2+]i and HSP-FRET to assess redox signaling in the same cells. Fluorescence images for the 2 sensors were alternated, and three sets of ratiometric measurements were collected every 20 seconds. This provided a virtually simultaneous assessment of calcium and redox responses in the same cells. Ratiometric images were acquired during baseline, followed by 30 minutes of hypoxia (Figure 7). This analysis revealed that the initial increases in ROS and [Ca2+]i could not be distinguished temporally. Although further increases in HSP-FRET ratios accumulated over time, these results do not refute the conclusion that the initial increase in calcium was triggered by ROS.
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Discussion |
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This study extends prior work by demonstrating that hypoxia increases oxidant signaling in the cytosol of cultured PASMCs during hypoxia. Using HSP-FRET in parallel with measurements of GSH/GSSG to assess the redox state of the cell, we observed a shift toward a more oxidized state in PASMCs during hypoxia. The increase in oxidant signaling was attenuated by the antioxidants PDTC and NAC, which act by reducing thiol groups and enhancing ROS scavenging. The oxidant signal was also attenuated by overexpression of GPx1, cytosolic catalase, or mitochondrial catalase, further supporting an involvement of H2O2. The relationship between ROS signaling and the increase in [Ca2+]i was explored using YC2.3, a FRET-based ratiometric Ca2+ sensor. Increases in [Ca2+]i during hypoxia were attenuated by antioxidants, as well as by overexpression of GPx1 or cytosolic or mitochondrial catalase. Myxothiazol, an inhibitor of electron transport at complex III, inhibited the responses to hypoxia, suggesting the mitochondrial ETC is the source of the hypoxia-induced ROS signal. Cyanide, which inhibits at complex IV, increased the Ca2+ signal but did not prevent the oxidant signal. These findings indicate that a fully functional ETC is not required for the hypoxic response but that electron flux through complex III is required because it is the source of the increase in ROS responsible for triggering the increase in [Ca2+]i during hypoxia in PASMCs. These findings support the hypothesis that mitochondria are required for O2 sensing in HPV because of their ability to augment ROS signaling during hypoxia.
Increased ROS Signaling Triggers HPV
Our results do not support the proposed model in which hypoxia decreases the generation of ROS in PASMCs and shifts the cytosol to a more reduced state.2,9–11,31,32 According to that scheme, the decrease in oxidant signaling causes closure of redox-sensitive Kv channels,2,11 resulting in membrane depolarization and the opening of voltage-gated Ca2+ channels.2,31 Central to the contrast between that model and ours is the issue of whether oxidant stress in PASMCs increases or decreases during hypoxia. In that regard, some investigators have detected increases,14–16 whereas other studies find decreases.2,9,10 Resolution of this issue has been hindered by the lack of a ratiometric sensor capable of detecting changes in the redox status of thiol-containing proteins in live cells. Using HSP-FRET, the conformation of which is regulated by a redox-sensitive HSP-33 domain, we now find evidence of protein thiol oxidation during hypoxia. Because the expression of HSP-FRET is predominantly within the cytosol, these findings indicate that oxidant signaling must occur in that compartment.
The hypoxia-induced increase in oxidant stress detected with HSP-FRET is consistent with the measured decrease in GSH/GSSG, although the latter responded more slowly than did the former, which began to change within minutes. It seems likely that endogenous glutathione reductase would defend against decreases in GSH/GSSG, thereby delaying the appearance of changes in that ratio. By contrast, refolding of HSP-FRET appears to occur slowly (data not shown), so changes in the oxidation state of HSP-FRET molecules could accumulate more rapidly and allow earlier detection. Both responses are consistent with the observation that chemical antioxidants and overexpression of oxidant scavenging proteins attenuate both the oxidant signal and the downstream calcium response to hypoxia. The involvement of a hypoxia-induced ROS signal is further supported by the observation that exogenous H2O2 mimics HPV in PASMCs and in isolated lungs.14,17
ROS Signaling and Increases in [Ca2+]i
Although ratiometric sensors such as Fura-2 are available for assessing [Ca2+]i, the accumulation of these probes in mitochondria and other organelles can interfere with their ability to provide a measure of Ca2+ in the cytosol.33 To address this limitation we used a Ca2+-sensitive, ratiometric FRET sensor (YC2.3) to measure changes in Ca2+ signaling in PASMCs.29 YC2.3 was expressed in the cytosol, and its property as a high-affinity sensor of Ca2+ permits assessment of signals in that compartment.27–29
Many studies have shown that PASMC contraction during hypoxia results from an increase in [Ca2+]i.3,4,6,8,31 The present study extends that work by showing a connection between hypoxia-induced increases in ROS signaling and [Ca2+]i in PASMCs. Both PDTC and NAC attenuated the hypoxia-induced increase in [Ca2+]i, as did overexpression of the antioxidant proteins GPx1 and catalase, consistent with previous studies.17 Mitochondria-targeted catalase also attenuated the response, which suggests that H2O2 arising from the mitochondria is important for triggering increases in [Ca2+]i during HPV. However, overexpression of catalase in the mitochondria could conceivably enhance the scavenging of oxidants originating in the cytosol, so this result should be interpreted with caution. We note that the pharmacological antioxidants were more effective at attenuating the responses than were the enzymatic antioxidants. This may be attributable to an ability of the pharmacological agents to access subcellular compartments that the protein antioxidants cannot reach. Moreover, pharmacological antioxidants can act both to scavenge ROS and to chemically reduce the cellular targets of those oxidant signals, producing a more effective attenuation of the downstream response than with ROS scavengers alone.
Overexpression of SOD-II enhanced the [Ca2+]i response to hypoxia. Because SOD-II expression is limited to the matrix compartment, these results strengthen the conclusion that H2O2 arising from that compartment contributes to HPV. However, Rodríguez et al observed that SOD-II overexpression in the mitochondrial matrix of HT-1080 fibrosarcoma cells caused an increase in H2O2 production through an unknown mechanism unrelated to its SOD activity.34 Such a paradoxical increase in oxidant production by SOD-II might explain why [Ca2+]i responses were augmented by overexpression of SOD-II in our study. Nevertheless, we did not find evidence of an elevated normoxic [Ca2+]i that might have been expected if basal levels of ROS production had been accelerated. In either case, our findings reveal that H2O2 arising from mitochondria contributes to the hypoxic response, consistent with previous reports showing that antioxidants block the response to hypoxia in intact lungs, in isolated PA vessels, and in PASMCs.14,16,17 Our results are not consistent with the conclusions by Olschewski et al, who showed that exogenous reducing or oxidizing agents caused contraction or relaxation of PA vessels in accordance with their model of decreased ROS production in hypoxia.12 The basis for these contradictory findings is unclear but may relate to the high concentrations of reducing or oxidizing agents used in their study, which may have affected multiple redox-sensitive targets in the cell.
Ours is the first study to compare the time course of ROS signaling (HSP-FRET) and the [Ca2+]i response (Fura-2) in the same PASMCs during hypoxia. Within the resolution limits of these methods, there was no detectable difference in the time at which both signals began to increase. Because one signal did not clearly increase before the other, these results do not reveal whether one signal triggers the other or not. However, they do demonstrate a clear temporal association between the signals, and they do not exclude the possibility that ROS may have triggered the initial increase in cytosolic calcium. The observation that antioxidant agents and targeted antioxidant proteins attenuate the ROS response and the calcium response indicates that ROS are required for the increase in [Ca2+]i during hypoxia in PASMCs.
Mitochondrial Electron Transport and the Source of ROS During Hypoxia
Previous studies suggest that HPV requires electron transport in the proximal but not the distal region of the ETC.14,17,35,36 For example, inhibition of complex I by rotenone or diphenylene iodonium abrogates HPV,14,17,36 whereas inhibitors acting at more distal sites in the ETC, such as cyanide or antimycin A, fail to inhibit the response. This indicates that a fully functional ETC is not required for HPV,14,17,35 and it supports the observation that hypoxia-induced changes in [ATP] do not mediate HPV.37 Myxothiazol inhibits the binding of ubiquinol at complex III, thereby preventing the oxidation of ubiquinol and the formation of ubisemiquinone. We find that myxothiazol attenuates the hypoxia-induced increase in oxidant stress and the associated increase in [Ca2+]i, which underscores the likely role of ubisemiquinone as a the source of electrons responsible for superoxide generation, H2O2 production, and the subsequent increase in calcium. An earlier study reported that myxothiazol induces H2O2 production in isolated heart mitochondria under normoxia,38 which complicates our interpretation of its effects on ROS generation. We did not detect an increase in ROS production with myxothiazol during normoxia, either in the form of a decrease in GSH/GSSG or by the HSP-FRET sensor. This may be attributable to differences in isolated mitochondria versus whole cells. Future studies with genetic tools will be useful in resolving this issue definitively. Cyanide had no effect on the hypoxia-induced increase in ROS, and it augmented the hypoxia-induced increase in [Ca2+]i. Because complex IV acts in series with complex III, one might expect cyanide to block electron transport through complex III by preventing electron flux from complex III to cytochrome c. Conceivably, electron leak pathways from cytochrome c to alternate targets, such as p66Shc,39 would permit continued superoxide production at complex III during cyanide treatment, thus maintaining ROS generation during hypoxia.40 That each of these mitochondrial inhibitors increases the cytosolic NAD(P)/NAD(P)H ratio, whereas only the proximal inhibitors block HPV, supports the view that changes in the NAD(P)/NAD(P)H couple are not responsible for signaling hypoxia in HPV hypoxia.31
The mechanism by which hypoxia augments ROS signaling from the electron transport chain under conditions of hypoxia remains unresolved. Three potential mechanisms have been proposed.41 The "Vectoral Transport" hypothesis suggests that hypoxia may increase the relative release of ROS from complex III toward the intermembrane space, while decreasing the relative release toward the matrix. The "Semiquinone Lifetime" hypothesis suggests that a decrease in O2 interaction with protein or lipids at complex III could prolong the lifetime of ubisemiquinone at complex III. Finally, the "Oxygen Access" hypothesis suggests that hypoxia might increase the access of O2 to semiquinone radical moiety at complex III. In each of these mechanisms, membrane O2 levels would affect lipid-protein structure so as to increase electron transfer from ubisemiquinone to O2, yielding an increase in superoxide release to the cytosol despite a decrease in the availability of oxygen.
Oxidation of HSP-FRET during hypoxia has recently been observed in other cell types, where hypoxia-induced, mitochondrial ROS signaling was shown to trigger stabilization of the hypoxia-inducible transcription factor (HIF).22,40 HPV is an acute response mediated by posttranslational mechanisms, yet it also involves an increase in the generation of ROS by the mitochondria. Collectively, these observations suggest that the mitochondrial oxygen sensor is capable of triggering a wide range of responses to hypoxia in diverse cell types. Conceivably, tissue-specific responses to hypoxia could be regulated by the expression of redox-regulated signaling molecules capable of activating the cell-specific responses to the same upstream oxidant release from the mitochondria. Future studies are required to fully address this hypothesis.
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Acknowledgments |
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We thank Dr Jingxiang Bai (Mount Sinai School of Medicine, New York) for providing recombinant adenoviruses containing Lac Z, catalase, and mitochondria-targeted catalase; Dr Christopher Rhodes (Pacific Northwest Research Institute, Seattle, Wash) for the adenovirus expressing YC2.3; and the University of Iowa Viral Vector Core for the adenoviruses expressing SOD-I, SOD-II, and GPx1. The HSP-FRET sensor was contributed by Drs Beatrice Hoyos and Ulrich Hammerling (Memorial Sloan-Kettering Cancer Center, New York). We also thank Dr Hong Chen and Chan Boriboun for technical assistance.
Sources of Funding
Supported by NIH grants HL66315, HL35440, and HL079650 (to P.T.S); American Heart Association Grant 0235457Z (to G.B.W.); and NIH/NIDDK DK63493 and a Research Grant from the American Diabetes Association (to M.W.R.).
Disclosures
None.
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Footnotes |
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Original received September 7, 2005; resubmission received August 14, 2006; revised resubmission received September 11, 2006; accepted September 14, 2006.
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References |
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