Department of Anatomy, School of Medicine, Case Western Reserve University, Cleveland, Ohio
Submitted 8 September 2004 ; accepted in final form 31 December 2004
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ABSTRACT |
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hypoxia-inducible factor 1; oxygen sensing
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MATERIALS AND METHODS |
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Plasmid constructs and cell transfection.
Plasmid constructs HIF-1 (521652)-green fluorescent protein (GFP)-V5 and HIF-1
(P402A/P564A)-V5 were generously provided by Dr. Thilo Hagen (University of Nottingham, UK) and were previously described (9). Briefly, HIF-1
(521652)-GFP-V5 was generated by fusing NH2 terminal residues 521652 of HIF-1
, which contains oxygen-dependent degradation domain of HIF-1
, to GFP-V5 in pcDNA3 vector (9). HIF-1
(P402A/P564A)-V5 was generated by replacing both Pro402 and Pro564 with alanines of the WT-HIF-1
in pcDNA3, which results in HIF-1
that is resistant to prolyl hydroxylase-mediated degradation (9). Cells were transfected with plasmid DNA using Lipofectamine (Invitrogen) reagent and allowed to recover for 24 h, followed by exposure to hypoxia and chemical treatment and harvest in cell lysis buffer for immunoblotting.
Immunoblot analysis.
To determine HIF-1 protein levels in cultured cells, either nuclear extracts (2) or whole cell lysates in radioimmunoprecipitation assay buffer [50 mM Tris·HCl (pH 7.6), 0.1% sodium dodecyl sulfate (SDS), 2 mM dithiotreitol, 0.5% Nonidet P-40] containing protease inhibitor cocktail (Roche Biologicals) were prepared, and proteins were subjected to SDS-PAGE electrophoresis. Membranes were blocked with 5% nonfat dry milk. For HIF-1
detection, either primary rabbit polyclonal anti-HIF-1
antibodies raised against COOH terminal peptide or Transduction Laboratories mouse monoclonal antibodies (Lexington, KY) were used, followed by secondary antibody detection by enhanced chemiluminescence (Amersham, Piscataway, NJ). Horseradish peroxidase-labeled anti-V5 antibody was purchased from Invitrogen (Carlsbad, CA).
Nuclear protein extract preparation. After treatment, cells were scraped into cold PBS, centrifuged, and washed in five packed cell volumes of buffer containing 10 mM Tris·HCl (pH 7.5), 1.5 mM MgCl2, 10 mM KCl, and freshly supplemented with dithiothreitol, sodium vanadate, PMSF, leupeptin, and aprotinin. This was followed by 10 min of incubation in the same buffer on ice and homogenization in a glass Dounce homogenizer. Nuclei were pelleted by centrifugation at 10,000 g for 10 min, the supernatant was discarded, and nuclei resuspended in a buffer [0.42 M KCl, 20 mM Tris·HCl (pH 7.5), 20% (vol/vol) glycerol, 1.5 mM MgCl2], freshly supplemented with dithiothreitol, sodium vanadate, PMSF, leupeptin, and aprotinin. The suspension was rotated at 4°C for 30 min and then centrifuged for 30 min at 14,000 rpm. The supernatant containing the nuclear proteins was collected.
ROS measurements in cells. Cells were cultured in 24-well plates to 8090% confluency and, before exposure, loaded with 10 µM 5- and 6-chloromethyl-2,7-dichlorodihydrofluorescein diacetate for 30 min in the culture medium without phenol red. Cellular esterases cleave the acetoxymethyl group, forming a charged molecule that becomes highly fluorescent when oxidized to dichlorofluorescein (DCF). Baseline DCF fluorescence was measured with a CytoFluor 4000 plate reader (450 nm excitation, 530 nm emission), followed by addition of inhibitors in the medium. Subsequently, the cells were placed in hypoxia chambers or kept in the normoxic incubator. After 3 h of incubation, DCF fluorescence was measured. All experiments were performed in quadruplicate.
Cell viability analysis. Cells were treated with oligomycin under either normoxic or anoxic conditions for 4 and 8 h, followed by cell viability assay using Trypan blue exclusion (0.4% Trypan blue, light microscopy).
ATP measurements in cells. ATP measurement in cells was performed as described previously (2). Briefly, cells were washed with PBS and lysed by the addition of 0.1 M NaOH. The cells and media were collected, rapidly frozen, and stored at 80°C. ATP intracellular concentrations were analyzed with a luciferase bioluminescence procedure (2). A total of 10 µl of cell extract were mixed with 50 µl of ATP reagent composed of 0.5 ml of imidazole-HCl buffer [1 M], 0.02 ml of MgCl2 [1 M], 0.750 ml of KCl [1 M], and vortexed. To measure ATP content, 10 µl of cell lysate were mixed with luciferin-luciferase reagent (250 µl) and the light emission was measured using a Lumi-Vette luminometer (model S900, Chrono-log, Havertown, PA). All experiments were performed in triplicate, and the ATP content was normalized for cell protein. The protein content was measured using a standard Bradford assay (Bio-Rad, Hercules, CA).
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RESULTS |
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Inhibitory effects of mitochondrial respiratory chain blockers on HIF-1 accumulation depends on O2 concentration.
HIF-1
protein levels vary proportionally over a physiologically relevant range of O2 tension. We investigated whether mitochondrial inhibitors are equally effective in preventing HIF-1
accumulation in cells under moderate hypoxia (1.5% O2) and severe hypoxia (anoxia). To achieve near-anoxic conditions, cells were exposed to a gas mixture (5% CO2-95% N2) that still contained residual 0.20.3% oxygen and designated here as anoxia. Respiratory chain inhibitors markedly prevented HIF-1
protein increase under hypoxic conditions (Fig. 3, A and B) but were less effective in inhibiting anoxic HIF-1
increase (Fig. 3, A and B). ROS generation is higher under hypoxic than anoxic conditions (26), and this has been used to explain differential effects of respiratory chain inhibitors on HIF-1
accumulation under hypoxic and anoxic conditions (26). However, our findings (Figs. 1 and 2) demonstrate that ROS is not involved in hypoxic HIF-1
regulation. Taken together, these results indicate that the molecular mechanism of differential effects of mitochondrial inhibitors on HIF-1
accumulation under hypoxic and anoxic conditions is distinct from mitochondrial ROS generation.
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Oligomycin prevents hypoxia-induced HIF-1 accumulation.
Mitochondrial respiratory chain constitutes part of a larger functional entity that is affected by coupling of electron transport and oxidative phosphorylation in living cells (21). This prompted us to investigate whether inhibition of F0F1-ATPase activity might affect HIF-1
regulation. We demonstrate that oligomycin, an F0F1-ATPase inhibitor, suppressed HIF-1
accumulation in hypoxic Mum2B (Fig. 4A) and U87 cells (Fig. 4E) without altering HIF-1
content during normoxia (Fig. 4D). Oligomycin did not affect HIF-1
upregulation by hypoxia-mimicking agents DFO and DMO (Fig. 4, B and C, respectively), which directly inhibit the prolyl hydroxylation step of HIF-1
(13, 14). To ascertain that cell response to hypoxia is not diminished by possible cytotoxic effects of oligomycin and that cells have retained the ability to upregulate HIF-1
, inhibitors of prolyl hydroxylase activity DFO and DMO were added to oligomycin-treated hypoxic cells (Fig. 5A). Both DFO and DMO reversed oligomycin-induced inhibition of HIF-1
in hypoxic cells, confirming that cells have retained the ability to activate normally HIF-1
response under these experimental conditions (Fig. 5A). Inhibition of F0F1-ATPase leads to a decrease in cellular ATP that affects energetic status of the cell; thus we monitored ATP changes caused by oligomycin under normoxic and anoxic conditions (Fig. 5B). Oligomycin did not affect ATP levels under normoxic conditions and had a minor effect under anoxic conditions (17% decrease in ATP after 3 h of combined anoxia plus oligomycin treatment compared with normoxic control; Fig. 5B). This is in agreement with the notion that tumor cells have high glycolytic capacity, and short-term decrease in oxidative phosphorylation does not markedly alter cellular ATP. This result indicates that changes in the cellular ATP are not the underlying cause of the effect of oligomycin on HIF-1
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Effect of oligomycin on HIF-1 accumulation is also dependent on O2 concentration.
The inhibitory effect of oligomycin on HIF-1
was also dependent on the degree of oxygen concentration; the effect was small under anoxic conditions and marked during hypoxia (1.5% O2) (Fig. 6). Treatment with oligomycin does not decrease, but rather increases, mitochondrial ROS production (21); hence, this result further supports the notion that inhibition of F0F1-ATP synthase prevents HIF-1
accumulation via a mechanism that is distinct from ROS generation during hypoxia.
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DISCUSSION |
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Earlier reports indicated that mitochondrial respiratory chain activity is required for accumulation of HIF-1 during hypoxia (6, 7), whereas others have not found evidence to support a conclusive role for mitochondria in regulating HIF-1
(30, 31). Inhibition of electron transport chain by rotenone and myxothiazol (1, 2, 6, 7, 9, 26) has been shown to prevent HIF-1
protein accumulation in hypoxic cells. Nitric oxide (NO) inhibits HIF-1
accumulation during hypoxia (2, 9, 18, 29); effects on mitochondrial complex I (2) as well as complex IV (9), have been implicated as underlying mechanisms. Notably, NO under certain conditions has been reported (20) to induce HIF-1
in normoxic cells (reviewed in Ref. 4) and possibly affect prolyl hydroxylase function. Here we show that inhibition of respiratory complex III by either myxothiazol or antimycin A (Fig. 2, A and B) and inhibition of complex IV also inhibited HIF-1
accumulation (Fig. 2, A and B), in agreement with the report by Hagen et al. (9), and that the use of antioxidants did not inhibit hypoxia-induced accumulation, although it inhibited cobalt-induced HIF-1
(Fig. 1A). In agreement with our findings, the inhibitory effect of antioxidants on cobalt-induced HIF-1
expression was recently reported by others (5). Interestingly, Chachami et al. (5) showed that the effect of cobalt on HIF-1
is mediated via phosphatidylinositol 3-kinase, rather than by interference of cobalt with iron availability for prolyl hydroxylase function, as suggested earlier (14). These results further support the notion that mitochondrial ROS does not regulate HIF-1
in hypoxic cells; instead, mechanisms other than complex III-generated ROS must account for inhibition of HIF-1
accumulation by respiratory chain inhibitors.
The mitochondrial respiratory chain constitutes part of a larger functional entity that is affected by coupling of electron transport and oxidative phosphorylation in living cells. Oligomycin, an inhibitor of F0F1-ATPase, also suppressed accumulation of HIF-1 in hypoxic tumor cells (Fig. 4A), and this effect is specific for oxygen deprivation because oligomycin had no effect on DFO- or DMO-induced HIF-1
(Fig. 4, B and C). Other respiratory chain inhibitors (rotenone, myxothiazol) similarly inhibit only hypoxia-induced HIF-1
expression, but not HIF-1
induction by hypoxia-mimicking agents DFO and cobalt (6, 7), further supporting a specific oxygen-dependent effect on HIF-1
regulation due to respiratory chain inhibition.
These findings, as well as previously reported results regarding HIF-1 inhibition by mitochondrial inhibitors (1, 6, 7, 9, 26), could be explained by the model suggested by Hagen et al. (9), based on the notion that inhibition of mitochondrial respiratory chain complexes I, III, and IV decrease oxygen consumption, resulting in redistribution of oxygen to cytosol, which in turn helps to maintain prolyl hydroxylase activity, resulting in HIF-1
protein degradation. Here we demonstrate that oligomycin causes destabilization of HIF-1
via a prolyl hydroxylase-dependent degradation (Fig. 7, A and B), and this could also be explained by the same model (9). Inhibition of ATPase would prevent the proton flux back through the mitochondrial inner membrane via the ATP synthase proton channel, keeping the electrochemical gradient at its maximum potential difference (21). This would inhibit proton pumping by complexes I, III, and IV, cause stalling of electron flow through the electron transport chain, and result in impaired oxygen consumption, which would explain the effect of oligomycin. Taken together, implications of these results as well as implications of the model proposed by Hagen et al. (9) would be that inhibition of respiratory chain during moderate hypoxia (1.5% O2) causes redistribution of oxygen to cytosol, thereby maintaining the activity of prolyl hydroxylases, a necessary step for HIF-1
protein degradation. However, under conditions of near-anoxia when oxygen concentration is nearly diminished, any significant oxygen redistribution is not possible. Consequently, inhibition of the respiratory chain under anoxic conditions should not be able to prevent hypoxic accumulation of HIF-1
. Indeed, our results show that HIF-1
accumulation during anoxia is relatively insensitive to respiratory chain inhibition, in contrast to hypoxia (Fig. 3, A and B; Fig. 6).
An alternative mechanism that could incorporate the observed effects of respiratory chain inhibitors on hypoxic HIF-1 regulation emerged from a recent study (8). Impaired electron transport might have a secondary effect on TCA cycle, resulting in changes in cellular concentration of intermediate products. Some TCA cycle intermediate products have the ability to affect prolyl hydroxylase activity and consequently HIF-1
accumulation, as shown in the study by Verma and colleagues (8). This appears intriguing, because it could reconcile the role of respiratory chain, TCA cycle, and prolyl hydroxylase function on HIF-1
expression.
Hypoxia is an important component of the tumor microenvironment and HIF-1 is considered to be a positive factor for tumor progression by virtue of stimulating tumor angiogenesis, mediating metabolic adaptations of tumor cells to hypoxia, and in many cases by increasing resistance to hypoxia-induced cell death (23, 27). In addition to implications for understanding mitochondrial mechanisms of HIF-1
regulation, the ability of oligomycin to interfere with HIF-1
regulation could have other potential applications. Recently, a family of F0F1-ATPase inhibitors, including oligomycin, was shown to have selective cytotoxic effects on human tumor cell lines (25). These agents selectively sensitized tumor cells to apoptosis (25) and characteristically altered gene expression profiles of the 60 human cancer cell lines of the National Cancer Institute (25). The exact mechanisms for induction of apoptosis by this strategy remain to be established. In this study, we chose to use two human cell lines that are highly resistant to apoptosis, Mum2B uveal melanoma and U87 gliobastoma, to investigate how oligomycin affects HIF-1
expression during short-term hypoxia. The results point toward an additional pathway through which agents like oligomycin might enhance tumor cell killing by preventing HIF-1
protein accumulation during hypoxia. This sets the stage for further investigation of the effects of this class of agents on sensitization of hypoxic tumor cells using as a model cells such as Mum2B and U87 that are very resistant to apoptosis. The ability of oligomycin to suppress HIF-1
during hypoxia suggests new possibilities to exploit this and other F0F1-ATPase inhibitory agents in designing new strategies aimed at increasing cytotoxic effects on tumor cells.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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