Oligomycin inhibits HIF-1{alpha} expression in hypoxic tumor cells

Yanqing Gong and Faton H. Agani

Department of Anatomy, School of Medicine, Case Western Reserve University, Cleveland, Ohio

Submitted 8 September 2004 ; accepted in final form 31 December 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Hypoxia-inducible factor-1 (HIF-1) is a key regulator of cellular responses to reduced oxygen availability. The contribution of mitochondria in regulation of HIF-1{alpha} in hypoxic cells has received recent attention. We demonstrate that inhibition of electron transport complexes I, III, and IV diminished hypoxic HIF-1{alpha} accumulation in different tumor cell lines. Hypoxia-induced HIF-1{alpha} accumulation was not prevented by the antioxidants Trolox and N-acetyl-cysteine. Oligomycin, inhibitor of F0F1-ATPase, prevented hypoxia-induced HIF-1{alpha} protein accumulation and had no effect on HIF-1{alpha} induction by hypoxia-mimicking agents desferrioxamine or dimethyloxalylglycine. The inhibitory effect of mitochondrial respiratory chain inhibitors and oligomycin on hypoxic HIF-1{alpha} content was pronounced in cells exposed to hypoxia (1.5% O2) but decreased markedly when cells were exposed to severe oxygen deprivation (anoxia). Taken together, these results do not support the role for mitochondrial reactive oxygen species in HIF-1{alpha} regulation, but rather suggest that inhibition of electron transport chain and impaired oxygen consumption affect HIF-1{alpha} accumulation in hypoxic cells indirectly via effects on prolyl hydroxylase function.

hypoxia-inducible factor 1; oxygen sensing


HYPOXIA-INDUCIBLE FACTOR-1 (HIF-1) plays a key role in regulating cellular responses to hypoxia in normal and tumor cells (reviewed in Refs. 17 and 27). HIF-1 is a heterodimeric protein that consists of two subunits, HIF-1{alpha} and HIF-1{beta} (28, 32, 33). HIF-1{beta}, identical to the aryl hydrocarbon receptor nuclear translocator, is a common binding subunit of many basic helix-loop-helix heterodimer transcription factors with Per-aryl hydrocarbon receptor nuclear translocator-Sim domains, besides HIF-1{alpha} (32). Under oxic conditions, HIF-1{alpha} protein is rapidly destabilized via ubiquitination and degradation by the proteasome (11, 12, 24). HIF-1{alpha} degradation is mediated by the von Hippel-Lindau protein (pVHL) (19), which binds directly to the oxygen-dependent degradation domain of HIF-1{alpha}, followed by ubiquitination and proteasome degradation (19). Interaction between pVHL and a specific domain of the HIF-1{alpha} subunit is regulated by a family of prolyl-4-hydroxylases (3, 10, 13, 14, 16, 34). Prolyl hydroxylases have a requirement for dioxygen, iron, ascorbate, and 2-oxoglutarate (13, 14). Under hypoxic conditions, due to the lack of oxygen, prolyl hydroxylation of the HIF-1{alpha} subunit is impaired and this leads to stabilization/accumulation of the protein. HIF-1{alpha} activation is, however, complex and subject to regulation by other signaling pathways and events (22, 27). Models based on a mitochondrial contribution to HIF-1{alpha} regulation during hypoxia have also been proposed (1, 2, 6, 7, 9, 18, 26); however, the contribution of mitochondria to HIF-1 regulation has been questioned by others (30, 31). Results of this study show that inhibition of electron transport complexes I, III, and IV, as well as inhibition of mitochondrial F0F1 ATPase, prevent HIF-1{alpha} expression and that mitochondrial reactive oxygen species (ROS) is not involved in HIF-1{alpha} regulation during hypoxia.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture and reagents. Human hepatoblastoma Hep3B [American Type Culture Collection (ATCC) HB-8064] and glioblastoma (ATCC HTB-14) cell lines were maintained in DMEM supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Mum2B human melanoma (kindly provided by Mary Hendrix, University of Iowa, Iowa City, IA) were grown in RPMI 1640 medium, supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. All media and antibiotics were purchased from Life Technologies. Cells were pretreated with vehicle control or indicated agents for 30 min before exposure to hypoxia (1.5% O2-5% CO2-93.5% N2) or anoxia (5% CO2-95% N2) in a Plexiglas modular chamber (Billups-Rothenberg, Del Mar, CA). Mitochondrial inhibitors, and N-acetyl-cysteine (NAC) were purchased from Sigma (St. Louis, MO), and Trolox was purchased from Calbiochem (La Jolla, CA).

Plasmid constructs and cell transfection. Plasmid constructs HIF-1{alpha} (521–652)-green fluorescent protein (GFP)-V5 and HIF-1{alpha}(P402A/P564A)-V5 were generously provided by Dr. Thilo Hagen (University of Nottingham, UK) and were previously described (9). Briefly, HIF-1{alpha}(521–652)-GFP-V5 was generated by fusing NH2 terminal residues 521–652 of HIF-1{alpha}, which contains oxygen-dependent degradation domain of HIF-1{alpha}, to GFP-V5 in pcDNA3 vector (9). HIF-1{alpha}(P402A/P564A)-V5 was generated by replacing both Pro402 and Pro564 with alanines of the WT-HIF-1{alpha} in pcDNA3, which results in HIF-1{alpha} 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{alpha} 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{alpha} detection, either primary rabbit polyclonal anti-HIF-1{alpha} 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 80–90% 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).


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Antioxidants do not prevent HIF-1{alpha} accumulation in hypoxic cells. To investigate the suggested role of mitochondria-derived ROS on HIF-1{alpha} accumulation during hypoxia (6, 7), cells were exposed to hypoxia in the presence or absence of antioxidants NAC and Trolox. HIF-1{alpha} accumulation in hypoxic Mum2B, Hep3B, and U87 cells was not affected by antioxidant treatment (Fig. 1A). To demonstrate cellular effects of Trolox, we investigated its effects on cobalt-induced HIF-1{alpha}. Figure 1A shows that Trolox effectively inhibited cobalt-induced HIF-1{alpha} in Mum2B cells. To confirm that the concentration of Trolox used in our experiments attenuated ROS production, DCF fluorescence was measured in cells exposed to normoxia or hypoxia. Hypoxia resulted in a 1.9-fold increase in ROS production, in agreement with other studies (7, 26) (Fig. 1B). Trolox dramatically decreased ROS production under either normoxic (89% decrease in DCF fluorescence) or hypoxic (97% decrease in DCF fluorescence) conditions (Fig. 1B), although it did not affect HIF-1{alpha} accumulation. These results suggested that mitochondrial ROS is not involved in HIF-1{alpha} regulation during hypoxia and prompted us to investigate further how inhibition of the respiratory chain downstream of the superoxide-generating site on complex III affects HIF-1{alpha} accumulation in hypoxic cells.



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 1. Lack of effect of antioxidants on hypoxia-inducible factor-1{alpha} (HIF-1{alpha}) accumulation during hypoxia. A: Mum2B, Hep3B, and U87 cells exposed to either normoxia, 1.5% O2 hypoxia (H), or 200 µM cobalt chloride (Co) for 4 h in the presence or absence of antioxidants N-acetylcysteine (Nac; 10 mM) and 1 mM Trolox (Tr). Whole cell lysates of Mum2B and U87 cells, and nuclear extracts of Hep3B cells were prepared and used to determine HIF-1{alpha} cellular content. B: inhibitory effect of 1 mM Trolox on reactive oxygen species (ROS) formation in Mum2B cells exposed to normoxia or 1.5% O2 hypoxia for 3 h determined by measuring dichlorofluorescein (DCF) fluorescence. Experiments were performed in quadruplicate, and results are expressed as DCF fluorescence units ± SD.

 
Inhibition of complex III with antimycin A and inhibition of complex IV prevents HIF-1{alpha} protein stabilization during hypoxia. It has been reported that inhibition of mitochondrial complex I (1, 2, 6, 7, 9) and complex III activity with myxothiazol (6, 7, 9), which blocks ROS generation from complex III during hypoxia, prevents hypoxic stabilization of HIF-1{alpha}. Here we show that in addition to myxothiazol, antimycin A also prevents an increase in HIF-1{alpha} protein during hypoxia (Fig. 2, A and B). The significance of complex III inhibition by these two inhibitors is based on the differential effect they have on ROS generated from complex III; upstream complex III inhibitor myxothiazol, but not downstream complex III inhibitor antimycin A, prevents ROS formation (6, 7). The ability of both inhibitors to prevent an increase in HIF-1{alpha} further supports the notion that ROS generated from complex III is not required for HIF-1{alpha} stabilization under hypoxic conditions.



View larger version (24K):
[in this window]
[in a new window]
 
Fig. 2. Effect of electron transport chain inhibitors on HIF-1{alpha} accumulation in hypoxic cells. A: Mum2B cells exposed to hypoxia (1.5% O2) for 3 h in the presence or absence of mitochondrial inhibitors: 0.5 µg/ml rotenone (Rot), 1 µM myxothiazol (Myx), 1 µg/ml antimycin A (AA), and 5 mM sodium azide (Az). Cell lysates were prepared and used to determine HIF-1{alpha} and actin levels (n = 2 independent experiments). B: U87 cells exposed to hypoxia (1.5% O2) for 3 h in the presence or absence of mitochondrial inhibitors: 1 µg/ml antimycin A and 5 mM sodium azide. Cell lysates were used to determine HIF-1{alpha} and actin levels (n = 2 independent experiments).

 
Hypoxic accumulation of HIF-1{alpha} was also prevented by azide, a complex IV inhibitor (Fig. 2, A and B). Together, these results show that inhibition of the respiratory chain at the level of complexes I, III, and IV prevents hypoxic accumulation of HIF-1{alpha} protein and that mechanisms other than complex III-generated ROS must account for HIF-1{alpha} inhibition in hypoxic cells.

Inhibitory effects of mitochondrial respiratory chain blockers on HIF-1{alpha} accumulation depends on O2 concentration. HIF-1{alpha} protein levels vary proportionally over a physiologically relevant range of O2 tension. We investigated whether mitochondrial inhibitors are equally effective in preventing HIF-1{alpha} 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 0.2–0.3% oxygen and designated here as anoxia. Respiratory chain inhibitors markedly prevented HIF-1{alpha} protein increase under hypoxic conditions (Fig. 3, A and B) but were less effective in inhibiting anoxic HIF-1{alpha} 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{alpha} accumulation under hypoxic and anoxic conditions (26). However, our findings (Figs. 1 and 2) demonstrate that ROS is not involved in hypoxic HIF-1{alpha} regulation. Taken together, these results indicate that the molecular mechanism of differential effects of mitochondrial inhibitors on HIF-1{alpha} accumulation under hypoxic and anoxic conditions is distinct from mitochondrial ROS generation.



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 3. Inhibition of HIF-1{alpha} accumulation by mitochondrial inhibitors depends on the cellular oxygen concentration. Mum2B cells (A) and U87 (B) cells exposed to either anoxia or 1.5% O2 hypoxia for 3 h in the presence or absence of 1 µg/ml antimycin A or 5 mM azide. Cell lysates were used to determine HIF-1{alpha} and actin levels. Scanning densitometry of autoradiogram signals for HIF-1{alpha} and actin was performed to calculate ratios of band intensities. Changes are expressed as percent decrease relative to HIF-1{alpha} maximum induction in anoxia or hypoxia (n = 2 independent experiments). C: ATP measurements in Mum2B cells treated with 1 µM myxothiazol, 1 µg/ml antimycin A, or 5 mM sodium azide for 3 h under normoxic and anoxic conditions. Cell lysates were used for ATP and total cellular protein measurements. Experiments were performed in triplicate, and results are expressed as means ± SD (nmol/mg protein).

 
Mitochondrial inhibitors did not affect significantly ATP levels under normoxic or anoxic conditions (Fig. 3C), consistent with the notion that tumor cells have high glycolytic capacity, and short-term decrease in oxidative phosphorylation did not alter cellular ATP. This indicates that potential changes in the cellular ATP are not the underlying cause of the effect of respiratory chain inhibitors on HIF-1{alpha}.

Oligomycin prevents hypoxia-induced HIF-1{alpha} 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{alpha} regulation. We demonstrate that oligomycin, an F0F1-ATPase inhibitor, suppressed HIF-1{alpha} accumulation in hypoxic Mum2B (Fig. 4A) and U87 cells (Fig. 4E) without altering HIF-1{alpha} content during normoxia (Fig. 4D). Oligomycin did not affect HIF-1{alpha} upregulation by hypoxia-mimicking agents DFO and DMO (Fig. 4, B and C, respectively), which directly inhibit the prolyl hydroxylation step of HIF-1{alpha} (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{alpha}, 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{alpha} in hypoxic cells, confirming that cells have retained the ability to activate normally HIF-1{alpha} 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{alpha}.



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 4. Oligomycin prevents hypoxia-induced HIF-1{alpha} accumulation but does not affect desferrioxamine (DFO)- or dimethyloxalyl glycine (DMO)-induced HIF-1{alpha}. A: Mum2B cells exposed to 1.5% O2 hypoxia (H) for 3 h in the absence or presence of 5 µg/ml (+) or 10 µg/ml (++) oligomycin (Oli). Whole cell lysates were used to determine HIF-1{alpha} and actin protein content. B: Mum2B cells were exposed to 1.5% O2 hypoxia or 200 µM DFO for 4 h in the presence or absence of 5 µg/ml of oligomycin. Cell lysates were prepared and used to determine HIF-1{alpha} and actin levels. C: Mum2B cells exposed to 1.5% O2 hypoxia for 3 h or 1 mM DMO for 4 h in the presence or absence of 5 µg/ml (+) or 10 µg/ml (++) oligomycin. Cell lysates were prepared and used to determine HIF-1{alpha} and actin levels. D: oligomycin does not affect baseline HIF-1{alpha} protein content. Mum2B cells were exposed to normoxia or 1.5% O2 hypoxia in the presence or absence of 5 µg/ml oligomycin. E: U87 cells were exposed to normoxia or 1.5% O2 hypoxia for 3 h in the absence or presence of 5 µg/ml oligomycin.

 


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 5. DFO and DMO reversed oligomycin suppression of HIF-1{alpha} in hypoxic cells. A: Mum2B cells were exposed to 1.5% O2 hypoxia for 4 h in the presence or absence of DMO (1 mM), DFO (200 µM), or oligomycin (5 µg/ml). Both DMO and DFO were able to reverse oligomycin suppression of HIF-1{alpha} induced by hypoxia. B: Mum2B cells were exposed to normoxia or anoxia for 3 h in the presence or absence of oligomycin (10 µg/ml). Cell lysates were used for ATP and total cellular protein measurements. Experiments were performed in triplicate, and results are expressed as means ± SD (nmol/mg protein).

 
To determine whether cell treatments resulted in any significant cytotoxicity, we performed a Trypan blue viability test. MUM2B and U87 cells did not show any significant decrease in viability during an 8-h exposure period to oligomycin under oxic, hypoxic, or anoxic conditions, significantly longer than the 3- to 4-h duration of our experiments (data not shown).

Effect of oligomycin on HIF-1{alpha} accumulation is also dependent on O2 concentration. The inhibitory effect of oligomycin on HIF-1{alpha} 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{alpha} accumulation via a mechanism that is distinct from ROS generation during hypoxia.



View larger version (47K):
[in this window]
[in a new window]
 
Fig. 6. Inhibitory effects of oligomycin on HIF-1{alpha} hypoxic induction depend on cellular oxygen concentration. Mum2B cells were exposed to normoxia, anoxia, or 1.5% O2 hypoxia for 3 h in the absence or presence of 5 µg/ml (+) or 10 µg/ml (++) oligomycin (Oli). Whole cell lysates were prepared to determine HIF-1{alpha} and actin protein content, followed by scanning densitometry of autoradiogram signals for HIF-1{alpha} and actin. Changes are expressed as percentage decrease relative to HIF-1{alpha} maximum induction in anoxia or hypoxia (n = 2 independent experiments).

 
To further investigate whether HIF-1{alpha} destabilization by oligomycin is dependent on the O2-dependent degradation domain of HIF-1{alpha} by a prolyl hydroxylase-dependent mechanism, we used a deletion construct containing residues 521 to 652 of HIF-1{alpha} (fused to GFP-V5 protein), which confers O2 dependence (9) (Fig. 7A). Immunoblotting with anti-V5 antibodies showed stabilization of the construct by hypoxia (1.5% O2) and destabilization in the presence of oligomycin (Fig. 7A). Cell treatment with DFO, used as a positive control, resulted in the stabilization of the deletion fragment as predicted. Therefore, inhibition of hypoxic HIF-1{alpha} accumulation by oligomycin is due to protein destabilization that involves a prolyl hydroxylase-dependent mechanism.



View larger version (28K):
[in this window]
[in a new window]
 
Fig. 7. Oligomycin-induced HIF-1{alpha} destabilization during hypoxia is dependent on prolyl hydroxylase activity. A: Mum2B cells were transfected with 5 µg of plasmid DNA of truncated construct HIF-1{alpha}(521–652)-GFP-V5, allowed to recover 24 h, and exposed to 1.5% O2 hypoxia in the presence or absence of oligomycin (10 µg/ml) for 3 h. Cell lysates were prepared, followed by SDS-PAGE and immunoblotting with anti-V5 antibody. DFO (200 µM) treatment was used as positive control. Actin bands indicate uniformity of loading. B: U87 cells were transfected with either 5 µg of wild-type (wt) HIF-1{alpha} plasmid or 5 µg of mutated mHIF-1{alpha} P402A/P564A-V5 (lane 7 contains untransfected control cell lysate). After 24 h of recovery, cells were exposed to 1.5% O2 hypoxia for 3 h in the presence or absence of 10 µg/ml oligomycin (Oli). Cell lysates were prepared, followed by SDS-PAGE and immunoblotting using anti-V5 antibody. Probing for actin indicates uniformity of loading.

 
We next compared the stability of full-length wild-type (WT) HIF-1{alpha} (WT-HIF-1{alpha}) and HIF-1{alpha} with two mutated prolines (HIF-P402A/P564A) that render HIF-1{alpha} resistant to prolyl hydroxylase-mediated degradation. Cells were exposed to hypoxia (1.5% O2) in the presence or absence of oligomycin (Fig. 7B). Hypoxia resulted in increased accumulation of WT-HIF-1{alpha} (Fig. 7B, lane 2), which was markedly reduced by oligomycin (Fig. 7B, lane 3). In contrast, HIF-P402A/P564A was expressed in normoxic cells (Fig. 7B, lane 4), confirming resistance to prolyl hydroxylase-mediated degradation, and expression was not increased during hypoxia (Fig. 7B, lane 5). Treatment with oligomycin did not cause any decrease in HIF-1{alpha} accumulation (Fig. 7B, lane 6). This result is consistent with the notion that oligomycin causes HIF-1{alpha} protein destabilization that is dependent on prolyl hydroxylation of Pro402 and Pro564.


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
HIF-1{alpha} cellular content is regulated by pVHL and prolyl hydroxylases, a group of enzymes that hydroxylate HIF-1{alpha} on specific proline residues (13, 14, 19). The regulation of HIF-1{alpha} expression is, however, more complex and dependent on other factors besides oxygen concentration. This is not surprising, considering that particular cell types in different tissues have different roles, perform different functions, and respond differently to various forms and degrees of stress and therefore have retained the ability to integrate multiple signals that converge into HIF-1{alpha} activation. There is evidence that glucose metabolism (15), tricarboxycyclic acid (TCA) cycle intermediates (8), and the function of the respiratory chain (1, 2, 6, 7, 9) affect HIF-1{alpha} regulation.

Earlier reports indicated that mitochondrial respiratory chain activity is required for accumulation of HIF-1{alpha} during hypoxia (6, 7), whereas others have not found evidence to support a conclusive role for mitochondria in regulating HIF-1{alpha} (30, 31). Inhibition of electron transport chain by rotenone and myxothiazol (1, 2, 6, 7, 9, 26) has been shown to prevent HIF-1{alpha} protein accumulation in hypoxic cells. Nitric oxide (NO) inhibits HIF-1{alpha} 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{alpha} 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{alpha} 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{alpha} (Fig. 1A). In agreement with our findings, the inhibitory effect of antioxidants on cobalt-induced HIF-1{alpha} expression was recently reported by others (5). Interestingly, Chachami et al. (5) showed that the effect of cobalt on HIF-1{alpha} 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{alpha} in hypoxic cells; instead, mechanisms other than complex III-generated ROS must account for inhibition of HIF-1{alpha} 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{alpha} 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{alpha} (Fig. 4, B and C). Other respiratory chain inhibitors (rotenone, myxothiazol) similarly inhibit only hypoxia-induced HIF-1{alpha} expression, but not HIF-1{alpha} induction by hypoxia-mimicking agents DFO and cobalt (6, 7), further supporting a specific oxygen-dependent effect on HIF-1{alpha} regulation due to respiratory chain inhibition.

These findings, as well as previously reported results regarding HIF-1{alpha} 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{alpha} protein degradation. Here we demonstrate that oligomycin causes destabilization of HIF-1{alpha} 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{alpha} 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{alpha}. Indeed, our results show that HIF-1{alpha} 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{alpha} 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{alpha} 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{alpha} expression.

Hypoxia is an important component of the tumor microenvironment and HIF-1{alpha} 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{alpha} regulation, the ability of oligomycin to interfere with HIF-1{alpha} 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{alpha} 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{alpha} 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{alpha} 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.


    GRANTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This work was supported by National Institutes of Health Grant NS-41309 (to F. H. Agani) and the Geriatric Research, Education and Clinical Center of the Louis Stokes Veterans Affairs Medical Center.


    ACKNOWLEDGMENTS
 
We thank Thilo Hagen (University of Nottingham, UK) for providing plasmid constructs, Lawrence Sayre and David Lust (Case Western Reserve University, Cleveland, OH) for dimethyloxalylglycine synthesis and help with ATP measurements, respectively, and Mary Hendrix (University of Iowa, Iowa City) for providing the Mum2B cell line. Helpful discussions and suggestions by Charles Hoppel (Case Western Reserve University, Cleveland, OH) are greatly appreciated.


    FOOTNOTES
 

Address for reprint requests and other correspondence: F. H. Agani, Dept. of Anatomy, School of Medicine, Case Western Reserve Univ., 10900 Euclid Ave., Cleveland, OH 44106 (E-mail: fxa5{at}po.cwru.edu)

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.


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
1. Agani FH, Pichiule P, Chavez JC, and LaManna JC. The role of mitochondria in the regulation of hypoxia-inducible factor 1 expression during hypoxia. J Biol Chem 275: 35863–35867, 2000.[Abstract/Free Full Text]

2. Agani FH, Puchowicz M, Chavez JC, Pichiule P, and LaManna J. Role of nitric oxide in the regulation of HIF-1{alpha} expression during hypoxia. Am J Physiol Cell Physiol 283: C178–C186, 2002.[Abstract/Free Full Text]

3. Bruick RK and McKnight SL. A conserved family of prolyl-4-hydroxylases that modify HIF. Science 294: 1337–1340, 2001.[Abstract/Free Full Text]

4. Brune B and Zhou J. The role of nitric oxide (NO) in stability regulation of hypoxia inducible factor-1{alpha}. Curr Med Chem 10: 845–855, 2003.[CrossRef][ISI][Medline]

5. Chachami G, Simos G, Hatziefthimiou A, Bonanou S, Molyvdas PA, and Paraskeva E. Cobalt induces HIF-1{alpha} expression in ASM cells by a ROS and PI3K dependent mechanism. Am J Respir Cell Mol Biol 31: 544–551, 2004.[Abstract/Free Full Text]

6. Chandel NS, Maltepe E, Goldwasser E, Mathieu CE, Simon MC, and Schumacker PT. Mitochondrial reactive oxygen species trigger hypoxia-induced transcription. Proc Natl Acad Sci USA 95: 11715–11720, 1998.[Abstract/Free Full Text]

7. Chandel NS, McClintock DS, Feliciano CE, Wood TM, Melendez JA, Rodriguez AM, and Schumacker PT. Reactive oxygen species generated at mitochondrial complex III stabilize hypoxia-inducible factor-1{alpha} during hypoxia: a mechanism of O2 sensing. J Biol Chem 275: 25130–25138, 2002.[CrossRef]

8. Dalgard CL, Lu H, Mohyeldin A, and Verma A. Endogenous 2-oxoacids differentially regulate expression of oxygen sensors. Biochem J 380: 419–424, 2004.[CrossRef][ISI][Medline]

9. Hagen T, Taylor CT, Lam F, and Moncada S. Redistribution of intracellular oxygen in hypoxia by nitric oxide: effect on HIF1{alpha}. Science 302: 1975–1978, 2003.[Abstract/Free Full Text]

10. Huang J, Zhao Q, Mooney SM, and Lee FS. Sequence determinants in hypoxia-inducible factor-1{alpha} for hydroxylation by the prolyl hydroxylases PHD1, PHD2, and PHD3. J Biol Chem 277: 39792–39800, 2002.[Abstract/Free Full Text]

11. Huang LE, Arany Z, Livingston DM, and Bunn HF. Activation of hypoxia-inducible transcription factor depends primarily upon redox-sensitive stabilization of its {alpha} subunit. J Biol Chem 271: 32253–32259, 1996.[Abstract/Free Full Text]

12. Huang LE, Gu J, Schau M, and Bunn HF. Regulation of hypoxia-inducible factor 1{alpha} is mediated by an O2-dependent degradation domain via the ubiquitin-proteasome pathway. Proc Natl Acad Sci USA 95: 7987–7992, 1998.[Abstract/Free Full Text]

13. Ivan M, Kondo K, Yang H, Kim W, Valiando J, Ohh M, Salic A, Asara JM, Lane WS, and Kaelin WG Jr. HIF{alpha}-targeted for VHL-mediated destruction by proline hydroxylation: implications for O2 sensing. Science 292: 464–468, 2001.[Abstract/Free Full Text]

14. Jaakkola P, Mole DR, Tian YM, Wilson MI, Gielbert J, Gaskell SJ, von Kriegsheim A, Hebestreit HF, Mukherji M, Schofield CJ, Maxwell PH, Pugh CW, and Ratcliffe PJ. Targeting of HIF-{alpha} to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation. Science 292: 468–472, 2001.[Abstract/Free Full Text]

15. Lu H, Forbes RA, and Verma A. HIF-1 activation by aerobic glycolysis implicates the Wartburg effect in carcinogenesis. J Biol Chem 277: 23111–23115, 2002.[Abstract/Free Full Text]

16. Masson N, Willam C, Maxwell PH, Pugh CW, and Ratcliffe PJ. Independent function of two destruction domains in hypoxia-inducible factor-{alpha} chains activated by prolyl hydroxylation. EMBO J 20: 5197–5206, 2001.[Abstract/Free Full Text]

17. Masson N and Ratcliffe PJ. HIF prolyl and asparaginyl hydroxylases in the biological response to intracellular O2 levels. J Cell Sci 116: 3041–3049, 2003.[Abstract/Free Full Text]

18. Mateo J, Garcia-Lecea M, Cadenas S, Hernandez C, and Moncada S. Regulation of hypoxia-inducible factor-1alpha by nitric oxide through mitochondria-dependent and -independent pathways. Biochem J 376: 537–544, 2003.[CrossRef][ISI][Medline]

19. Maxwell PH, Wiesener MS, Chang GW, Clifford SC, Vaux EC, Cockman ME, Wykoff CC, Pugh CW, Maher ER, and Ratcliffe PJ. The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis. Nature 399: 271–275, 1999.[CrossRef][ISI][Medline]

20. Metzen E, Zhou J, Jelkmann W, Fandrey J, and Brune B. Nitric oxide impairs normoxic degradation of HIF-1{alpha} by inhibition of prolyl hydroxylases. Mol Biol Cell 14: 3470–3481, 2003.[Abstract/Free Full Text]

21. Nicholls DG and Ferguson S. The ATP synthase. In: Bioenergetics 3. San Diego, CA: Academic, 2002, p. 195–216.

22. Richard DE, Berra E, Gothie E, Roux D, and Pouyssegur J. p42/p44 mitogen-activated protein kinases phosphorylate hypoxia-inducible factor 1{alpha} (HIF-1{alpha}) and enhance the transcriptional activity of HIF-1. J Biol Chem 274: 32631–32637, 1999.[Abstract/Free Full Text]

23. Ryan HE, Lo J, and Johnson RS. HIF1{alpha} is required for solid tumor formation and embryonic vascularization. EMBO J 17: 3005–3015, 1998.[Abstract/Free Full Text]

24. Salceda S and Caro J. Hypoxia-inducible factor 1{alpha} (HIF-1{alpha}) protein is rapidly degraded by the ubiquitin-proteasome system under normoxic conditions. Its stabilization by hypoxia depends on redox-induced changes. J Biol Chem 272: 22642–22647, 1997.[Abstract/Free Full Text]

25. Salomon AR, Voehringer DW, Herzenberg LA, and Khosla C. Understanding and exploiting the mechanistic basis for selectivity of polyketide inhibitors of F0F1-ATPase. Proc Natl Acad Sci USA 97: 14766–14771, 2000.[Abstract/Free Full Text]

26. Schroedl C, McClintock DS, Budinger GR, and Chandel NS. Hypoxic but not anoxic stabilization of HIF-1{alpha} requires mitochondrial reactive oxygen species. Am J Physiol Lung Cell Mol Physiol 283: L922–L931, 2002.[Abstract/Free Full Text]

27. Semenza GL. HIF-1 and tumor progression: pathophysiology and therapeutics. Trends Mol Med 8: S62–S67, 2002.[CrossRef][ISI][Medline]

28. Semenza GL and Wang GL. A nuclear factor induced by hypoxia via de novo protein synthesis binds to the human erythropoietin gene enhancer at a site required for transcriptional activation. Mol Cell Biol 12: 5447–5454, 1992.[Abstract]

29. Sogawa K, Numayama-Tsuruta K, Ema M, Abe M, Abe H, and Fujii-Kuriyama Y. Inhibition of hypoxia-inducible factor 1 activity by nitric oxide donors in hypoxia. Proc Natl Acad Sci USA 95: 7368–7373, 1998.[Abstract/Free Full Text]

30. Srinivas V, Leshchinsky I, Sang N, King MP, Minchenko A, and Caro J. Oxygen sensing and HIF-1 activation does not require an active mitochondrial respiratory chain electron-transfer pathway. J Biol Chem 276: 21995–21998, 2001.[Abstract/Free Full Text]

31. Vaux EC, Metzen E, Yeates KM, and Ratcliffe PJ. Regulation of hypoxia-inducible factor is preserved in the absence of a functioning mitochondrial respiratory chain. Blood 198: 296–302, 2001.[CrossRef]

32. Wang GL, Jiang BH, Rue EA, and Semenza GL. Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension. Proc Natl Acad Sci USA 92: 5510–5514, 1995.[Abstract/Free Full Text]

33. Wang GL and Semenza GL. Purification and characterization of hypoxia-inducible factor 1. J Biol Chem 270: 1230–1237, 1995.[Abstract/Free Full Text]

34. Yu F, White SB, Zhao Q, and Lee FS. HIF1{alpha} binding to VHL is regulated by stimulus-sensitive proline hydroxylation. Proc Natl Acad Sci USA 98: 9630–9635, 2001.[Abstract/Free Full Text]