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Am J Physiol Cell Physiol 294: C1305-C1312, 2008. First published March 19, 2008; doi:10.1152/ajpcell.00007.2008
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CELLULAR AND MITOCHONDRIAL METABOLISM

Induction of HIF-2{alpha} is dependent on mitochondrial O2 consumption in an O2-sensitive adrenomedullary chromaffin cell line

Stephen T. Brown and Colin A. Nurse

Department of Biology, McMaster University, Hamilton, Ontario, Canada

Submitted 8 January 2008 ; accepted in final form 11 March 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
During low O2 (hypoxia), hypoxia-inducible factor (HIF)-{alpha} is stabilized and translocates to the nucleus, where it regulates genes critical for survival and/or adaptation in low O2. While it appears that mitochondria play a critical role in HIF induction, controversy surrounds the underlying mechanism(s). To address this, we monitored HIF-2{alpha} expression and oxygen consumption in an O2-sensitive immortalized rat adrenomedullary chromaffin (MAH) cell line. Hypoxia (2–8% O2) caused a concentration- and time-dependent increase in HIF-2{alpha} induction, which was blocked in MAH cells with either RNA interference knockdown of the Rieske Fe-S protein, a component of complex III, or knockdown of cytochrome-c oxidase subunit of complex IV, or defective mitochondrial DNA ({rho}0 cells). Additionally, pharmacological inhibitors of mitochondrial complexes I, III, IV, i.e., rotenone (1 µM), myxothiazol (1 µM), antimycin A (1 µg/ml), and cyanide (1 mM), blocked HIF-2{alpha} induction in control MAH cells. Interestingly, the inhibitory effects of the mitochondrial inhibitors were dependent on O2 concentration such that at moderate-to-severe hypoxia (6% O2), HIF-2{alpha} induction was blocked by low inhibitor concentrations that were ineffective at more severe hypoxia (2% O2). Manipulation of the levels of reactive oxygen species (ROS) had no effect on HIF-2{alpha} induction. These data suggest that in this O2-sensitive cell line, mitochondrial O2 consumption, rather than changes in ROS, regulates HIF-2{alpha} during hypoxia.

reactive oxygen species; hypoxia-inducible factor; mitochondrial inhibition; RNA interference

OXYGEN PLAYS A CRITICAL ROLE in cell survival, most importantly in ATP production as the terminal electron receptor during mitochondrial oxidative phosphorylation. As such, various mechanisms have evolved for detecting a fall in the partial pressure of oxygen (hypoxia), and these result in the initiation of adaptive responses, some of which are crucial for survival of the organism. One important mechanism involves activation of the transcription factor, hypoxia-inducible factor (HIF). Hypoxia-inducible factors are a family of transcription factors including HIF-1{alpha}, HIF-2{alpha}, and HIF-3{alpha} that are key regulators of gene expression induced by hypoxia (27). The functional complex is a heterodimer consisting of an {alpha}- and a β-subunit whose NH2 termini contain a basic helix-loop-helix domain responsible for dimerization and DNA binding. While both subunits are constitutively expressed, the {alpha}-subunit is rapidly degraded under normal oxygen conditions (normoxia) and is therefore unable to dimerize with HIF-1β subunit (17, 32). This degradation is dependent on the partial pressure of oxygen (PO2) such that the lower the PO2, the longer the half-life of HIF-1{alpha} (18). Increased stability under hypoxic conditions allows the subunits to dimerize and regulate transcription of a host of hypoxia-regulated genes (12, 26). Under normoxia, the HIF-{alpha} subunit undergoes an O2-dependent hydroxylation at proline residues 564 and 402 via three oxygenases termed prolyl hydroxylase domain enzyme (PHD)-1, PHD-2, and PHD-3 (22). When hydroxylated, these proline residues serve as binding sites for the von Hippel-Lindau tumor suppressor protein, which acts as the recognition unit for the E3 ubiquitin ligase complex and allows for HIF-{alpha} degradation (23).

Although considerable research has been carried out on HIF-{alpha} induction during hypoxia, the mechanism by which reduced PO2 leads to HIF-{alpha} accumulation still remains contentious. Given the central role of mitochondria as the cell's major O2 consumers, it is perhaps not surprising that many studies have identified the importance of the mitochondrial electron transport chain (ETC) in HIF-1{alpha} and HIF-2{alpha} induction during hypoxia (2, 5, 8, 9, 15, 16, 20, 21). Indeed, cells that lack functional mitochondria due to defective mitochondrial DNA ({rho}0 cells) fail to induce HIF-{alpha} during hypoxia but nevertheless do so under anoxia when prolyl hydroxylation is inhibited (1, 2, 9, 29, 31). Moreover, pharmacological inhibition of the mitochondrial ETC using complex I and complex III blockers, e.g., rotenone and myxothiazol, respectively, impairs HIF-{alpha} induction during hypoxia. This evidence has led to a model whereby increased production of reactive oxygen species (ROS) at the Qo site of complex III leads to inhibition of the PHDs during hypoxia, reduction in prolyl hydroxylation, and, consequently, HIF-{alpha} accumulation (2). This model is supported by experiments showing that exogenous application of ROS can induce HIF-{alpha} under normoxic conditions and that application of ROS scavengers can block hypoxic induction of HIF-{alpha} (15, 20). Because the major site of mitochondrial ROS production is thought to be the Qo site of complex III, inhibition of the ETC downstream of the Qo site should have no effect on HIF-{alpha} induction according to this model. While this prediction has been borne out in some studies (2, 9, 18), the opposite conclusion has been reached in others, thereby precluding a unifying theory on the role of mitochondria in HIF induction during hypoxia. For example, in studies on the human HEK-293 (16) and osteosarcoma (10) cell lines, the more distal ETC inhibitors including antimycin A (an inhibitor of complex III at a more distal site than myxothiazol) and cyanide or azide (complex IV inhibitors) blocked HIF-1{alpha} induction during hypoxia. In both studies, manipulation of ROS levels was without effect, leading the authors to propose an alternative model whereby oxygen consumption by the mitochondria generates a gradient that limits oxygen availability to the PHDs, thereby restricting prolyl hydroxylation and allowing for HIF-{alpha} stabilization. According to this scheme, general inhibition of the mitochondrial ETC causes a reduction in this gradient and an increase in oxygen availability for the PHDs, resulting in HIF-{alpha} degradation.

The above discrepancies on the role of mitochondria in HIF-1{alpha} induction during hypoxia may be attributable to the use of different cell lines. While it is believed that most cells have the ability to sense oxygen, certain cell types have evolved into specialized O2 sensors, including carotid body type I cells, pulmonary vascular smooth muscle cells, neuroepithelial body cells, and neonatal adrenal chromaffin cells (33). Such specialized O2 sensors have received relatively little attention in studies of the mechanisms of HIF induction. In the present study, we address this void by investigating the mechanisms of HIF-2{alpha} induction in a v-myc immortalized adrenal chromaffin cell line (MAH cells), which possesses several of the acute O2-sensing properties of their primary neonatal counterparts (11). The latter cells play a critical role in aiding survival of the newborn by directly sensing low PO2 during the birthing process and releasing catecholamines that mediate key physiological responses (25, 28). Our findings indicate that while functional mitochondrial are indeed required for HIF-2{alpha} induction in MAH cells, the underlying mechanisms appear to involve regulation of cellular O2 distribution rather than alteration in ROS levels.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture. MAH cells were grown in modified L-15/CO2 medium supplemented with 1% penicillin/streptomycin, 0.6% glucose, 10% fetal bovine serum, and 5 µM dexamethasone. Cells were incubated in a humidified atmosphere of 95% air-5% CO2 at 37°C and were passaged every 3 days. The cell suspension was centrifuged at 12,000 rpm for 2 min, the supernatant removed, and the pellet resuspended in fresh medium. Cells were plated on tissue culture dishes coated with poly-D-lysine and laminin. Exposure to chronic hypoxia was achieved by incubating the cells in a 37°C O2-CO2 incubator in a humidified atmosphere at variable O2 tensions.

Western blot analysis. Culture dishes were removed from the incubator and immediately placed on ice and lysed in buffer A (10 mM HEPES pH 7.6, 10 mM KCl, 0.1 mM EDTA pH 8, 0.1 mM EGTA pH 8, and 1 mM DTT) containing protein inhibitors (Complete Mini; Roche, Laval, Quebec, Canada). Cells were scraped off into a microfuge tube and incubated on ice for 15 min with intermittent vortexing. NP40 was then added to a final concentration of 0.6% and was vortexed for 1 min. The lysate was centrifuged at 13,000 rpm for 30 s, the supernatant removed, and the pellet resuspended in 50 µl of buffer C (20 mM HEPES pH 7.6, 0.4 M NaCl, 1 mM EDTA pH 8, 1 mM EGTA pH 8, 1 mM DTT, and 5% glycerol) containing protein inhibitors, before freezing at –80°C. The lysate was thawed on ice and centrifuged at 13,000 RPM for 5 min, and the resulting supernatant was removed and quantified using a Bradford assay. Twenty micrograms of nuclear-extracted protein was run on an 8% SDS-polyacrylamide gel at 120 V for 2 h. Protein was transferred from the gel onto a polyvinylidene difluoride membrane (Millipore, Bellerica, MA) and was incubated in either 1:1,000 dilution of HIF-2{alpha} (Novus Biologicals, Littleton, CO) rabbit polyclonal antibody or 1:2,000 dilution of Tata-binding protein (Santa Cruz Biotechnology, Santa Cruz, CA) rabbit polyclonal antibody at 4°C overnight. The membrane was washed four times with 1x PBS for 10 min each and was then incubated for 1 h at room temperature with 1:25,000 dilution of goat anti-rabbit horseradish peroxidase (HRP) antibody (Jackson Labs, Bar Harbour, ME). The membrane was washed four times in 1x PBS for 10 min each, and the blot was visualized using Immobilon Western Chemiluminescent HRP substrate (Millipore) and autoradiography. Western blot analysis was performed at least three times, and a representative blot is shown.

Generation and characterization of {rho}0 MAH cells. The {rho}0 cells were generated by the ethidium bromide method, as previously described by Buttigieg et al. (6). Mitochondrial DNA levels were examined and compared with control ({rho}+) cells grown in media in the absence of EtBr, pyruvate, and uridine. DNA was extracted from the cells using the QIAamp DNA mini Kit (Qiagen, Valencia, CA) following the manufacturer's protocol. DNA was quantified using an Eppendorf Biophotometer (Westbury, NY). Quantitative PCR was carried out on 500 ng of DNA using a MX3000P machine (Stratagene, La Jolla, CA) and the Absolute QPCR SYBR Green Mix (ABgene, Epson, UK). Gene-specific primers were designed using GeneFisher and synthesized by The Central Facility of the Institute for Molecular Biology and Biotechnology (MOBIX; McMaster University, Hamilton, ON, Canada) (14). The following primers were used and are listed as gene amplified and sequence (forward, reverse): β-actin: 5'-CCTAGTCGTTCGTCCTCATGC-3'and 5'-GAAGATCCTGACCGAGCGTG-3'; cox 1: 5'- TGGAGCCTGAGCAGGAATAG-3' and 5'-AATCTACGGATACCCCAGCA-3'. Verification of the PCR products was done using the QIAquick Gel Extraction kit (Qiagen) to extract PCR fragments from a 2% agarose gel. The DNA sample was then sequenced (at MOBIX). The sequencing results were analyzed by Basic Local Alignment Search Tool (BLAST), and the sequences were matched to the Rattus norvegicus cox 1 gene (GenBank accession number J01435) and β-actin (GenBank accession number XM_226922.4).

RNA interference transfection of MAH cells. Oligonucleotides containing the short hairpin RNA interference (RNAi) sequence for the Rieske iron-sulfur polypeptide (RISP; 5'-CTA TCG CCG TGC TGA AG TTT TCA AGA GAA ACT TCA GCA CGG CGA TAG-3'), cytochrome-c oxidase (COX10; 5'-TCA GGA ATG TCA CTA ATC ATT CAA GAG ATG ATT AGT GAC ATT CCT GA-3') and a scrambled negative control (scControl; 5'- TAG CGA CTA AAC ACA TCA ATT CAA GAG ATT GAT GTG TTT AGT CGC TA-3') were cloned into the pSuper Retroviral vector (4). The resulting plasmids were transfected into the phoenix packaging cell line and selected under puromycin for 1–2 wk. Subsequently, cell culture medium containing virus was collected and filtered, and the resulting viral supernatant was used to infect dividing MAH cells.

Quantitative RT-PCR. RNA from chromaffin cell cultures was extracted using the RNeasy Mini Kit (Qiagen) following the manufacturer's protocol. RNA was quantified in an Eppendorf Biophotometer, and 500 ng was treated with DNase I (Invitrogen, Carlsbad, CA) to remove any contaminating DNA. Reverse transcription was carried out on 100 ng of DNase-treated RNA using Superscript III (Invitrogen) and random primers (100 ng). A no-RT control was also run to test for the presence of DNA contamination (data not shown). Quantitative PCR was carried out using the Absolute QPCR SYBR Green Mix and a Stratagene MX3000P machine. Analysis was done with Stratagene MX3000p software using the {Delta}{Delta}Ct method (where Ct is cycle threshold). Gene-specific primers were designed using GeneFisher and were synthesized by MOBIX (14). The following primers were used and listed as gene amplified, sequence (forward, reverse), and annealing temperature: Lamin A/C: 5'-GCAGTACAAGAAGGAGCTA-3' and 5'-CAGCAATTCCTGGTACTCA-3', 55°C; RISP: 5'-CCA CAG TGG GCC TGA ATG TT-3' and 5'-AGC GTA TGC AAC ACC CAC AGT-3', 55°C; COX10: 5'-GCG TCC CCG CAC ACT ATT T-3' and 5'-GCT CAA GTG CTG AAC CGT GAC-3', 55°C. Verification of the PCR products was done using the QIAquick Gel Extraction kit (Qiagen) to extract PCR fragments from a 2% agarose gel. The DNA sample was then sequenced (at MOBIX) using an ABI Prism automated Sequencer (with T7 polymerase). The sequencing results were analyzed by BLAST, and the sequences were matched to the Rattus norvegicus Lamin A/C (GenBank accession number BC062018.1 and X99257.1), RISP (GenBank accession number NM_001008888), and COX10 (GenBank accession number XM_001079869).

Oxygen consumption assay. Oxygen consumption assays were performed using the BD oxygen biosensor system (BD Biosciences, San Jose, CA). Cells were seeded into the fluorescent dye-embedded 96-well microplate of the BD oxygen biosensor system at a density of 5 x 105 cells/well. Data were acquired at 1-min intervals over a period of 45 min with a heated fluorescent microplate reader. The rate of oxygen consumption was calculated and compared with control cells and expressed as a percentage relative to control.

Chemicals. EtBr, pyruvate, uridine, rotenone, myxothiazol, antimycin A, sodium cyanide, carboxy-PTIO potassium salt (CPTIO), ascorbic acid, Trolox, 2,3-dimethoxy-1,4-naphtoquinone, N-acetyl-L-cysteine, H2O2, tertbutyl peroxide, and desferrioxamine mesylate (DFX) were purchased from Sigma-Aldrich (St. Louis, MO). Dimethyloxaloylglycine was purchased from Biomol (Plymouth Meeting, PA).

Statistical analysis. Results are expressed as means ± SE, and statistical comparisons were made using nonparametric tests (Mann-Whitney U-test) as appropriate.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
HIF-2{alpha} induction under varying degrees of hypoxia. HIF-2{alpha} has been shown to be highly expressed in chromaffin cells in the organ of Zuckerkand and is critical for proper function during embryogenesis (30). In MAH cells, HIF-2{alpha} appeared to be the predominant form of HIF expressed, and hypoxia (2–10% O2) caused a concentration- and time-dependent induction of HIF-2{alpha} protein. As illustrated in Fig. 1A, HIF-2{alpha} protein was detectable at O2 concentrations between 8% and 2% during a 2-h exposure. At the most severe level of hypoxia (2% O2) tested, low levels of HIF-2{alpha} protein could be detected as early as 15 min after exposure, and the peak occurred after 1–2 h (Fig. 1B). Thus, HIF-2{alpha} induction in MAH cells is rapid, and the magnitude of the effect is both time and O2-concentration dependent.


Figure 1
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  		Fig. 1. Effects of hypoxia on hypoxia-inducible factor (HIF)-2{alpha} levels in MAH and {rho}0 cells. A: cells were exposed to normoxia (Nox, 21% O2) and to increasing levels of hypoxia (Hox; 10%, 8%, 6%, 4%, and 2% O2) for 2 h, and HIF-2{alpha} levels were probed (top). HIF-2{alpha} induction was observed at levels of 8% O2 and lower. B: cells were exposed to normoxia (21% O2) and hypoxia (2% O2) for 0.25, 0.50, 0.75, 1, and 2 h, and HIF-2{alpha} levels were probed (top). HIF-2{alpha} stabilization can be detected within 15 min of hypoxia. C: agarose gel of PCR products from DNA extracted from mitochondria-deficient ({rho}0) and control MAH cells using primers specific for β-actin and for the mitochondrial-encoded gene cox 1. The {rho}0 cells lacked the mitochondrial DNA for the cox 1 gene. Data are means ± SE; n = 3. *P < 0.01 vs. control. D: control (Cont) and {rho}0 MAH cells were treated with normoxia, hypoxia (2% O2), and dimethyloxaloylglycine (DMOG; 1 mM) for 2 h (top) and were probed for HIF-2{alpha} levels. Hypoxia failed to induce HIF-2{alpha} in the {rho}0 cells, although HIF-2{alpha} was still induced by DMOG. E: hypoxia failed to induce HIF-2{alpha} in the {rho}0 cells, although HIF-2{alpha} was induced by desferrioxamine mesylate (DFX). Tata binding protein (TBP) was used as an internal loading control.

 
Failure of HIF-2{alpha} induction in MAH cells lacking functional mitochondria. MAH cells lacking functional mitochondria ({rho}0 cells) were generated using the ethidium bromide technique (see MATERIALS AND METHODS). To confirm the {rho}0 status, DNA was extracted and quantitative PCR was performed for detection of the mitochondrial encoded gene cox 1 and the genomic encoded gene β-actin. The {rho}0 cells showed a loss of cox 1 but had normal levels of β-actin compared with the untreated control cells (Fig. 1C). To test whether or not HIF-2{alpha} induction was dependent on functional mitochondria, {rho}0 MAH cells were exposed to hypoxia (2% O2) for 2 h, a treatment that caused maximum induction in control cells (Fig. 1B). In agreement with previous studies on other cell types, whereas control MAH cells showed normal induction of HIF-2{alpha}, {rho}0 cells failed to show HIF-2{alpha} induction under hypoxia (Fig. 1D). The failure of hypoxia to induce HIF-2{alpha} in {rho}0 cells was due to the loss of mitochondrial function and not to an impairment of the HIF pathway, because {rho}0 cells did show HIF-2{alpha} accumulation when treated with the prolyl hydroxylase inhibitor dimethyloxaloylglycine (1 mM) or the iron chelator DFX (100 µM) for 4 h (Fig. 1, D and E).

Effects of specific RNAi knockdown and pharmacological inhibition of mitochondrial protein complexes on HIF-2{alpha} induction. There are conflicting reports on the effects of mitochondrial inhibitors on HIF induction in different cell types. In general, inhibitors that act upstream at mitochondrial complexes I and III, e.g., rotenone and myxothiazol, respectively, were found to inhibit HIF-{alpha} induction during hypoxia. However, inhibitors that acted at more distal regions and downstream of the myxothiazol binding site at complex III had varied effects. For example, Doege et al. (10) and Hagen et al. (16) found that all mitochondrial blockers, including the distal blocker of complex III, antimycin A, inhibited HIF-1{alpha} induction during hypoxia, whereas other groups reported that these inhibitors had no effect (2, 9, 18). To avoid total reliance on such drugs with questionable specificity, we used a genetic approach to perturb specific components of the mitochondrial ETC. Thus, RNAi was used to knock down the complex III subunit RISP and the complex IV subunit COX10. The RISP subunit has previously been shown to be critical for mitochondrial function, and mutations in COX10 have been associated with many metabolic disorders, illustrating their importance in ETC function (3, 15). MAH cells were infected with retrovirus containing an expression cassette for short hairpin RNAi molecules targeting RISP (shRISP), COX10 (shCOX10), and a scrambled control (scControl) (4). The amount of RISP and COX10 knockdown was assessed using quantitative RT-PCR, with lamin mRNA as control (Fig. 2A). Compared with the scrambled control, RISP and COX10 mRNA levels were knocked down by ~78% and ~83%, respectively. Under hypoxia (2% O2, 2 h), mutant MAH cells expressing shRISP and shCOX10 failed to induce HIF-2{alpha}; however, DFX treatment (100 µM, 4 h) was still effective in inducing HIF-2{alpha} (Fig. 2B).


Figure 2
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  		Fig. 2. Selective knockdown of complex III and IV prevents HIF-2{alpha} accumulation during hypoxia. A: Rieske Fe-S protein (RISP) and cytochrome-c oxidase subunit (COX10) mRNA levels were measured in MAH cells expressing scrambled control (scControl), RISP (shRISP), or cytochrome-c oxidase (shCOX10) short hairpin RNA (shRNA) molecules using quantitative RT-PCR. Results were normalized to Lamin A/C and are expressed as relative fold change compared with control. Data are means ± SE; n = 3. *P < 0.05 vs. control. B: HIF-2{alpha} protein levels were measured in cells expressing either scControl, shRISP, or shCOX10 shRNA molecules under normoxia, hypoxia (2% O2, 2 h), or cells treated with DFX (100 µM, 4 h). Knockdown of RISP and COX10 in MAH cells inhibited HIF-2{alpha} accumulation during hypoxia, but DFX treatment had no effect.

 
In light of these data, we compared the effects of various pharmacological ETC inhibitors, including the complex I inhibitor rotenone (1 µM), the proximal and distal complex III inhibitors myxothiazol (1 µM) and antimycin A (1 µg/ml), and the complex IV inhibitor cyanide (1 mM), on HIF-2{alpha} induction. In control MAH cells, all mitochondrial inhibitors blocked HIF-2{alpha} induction during hypoxia (2% O2, 2 h; Fig. 3A). Notably, however, they all failed to block DFX (100 µM, 4 h) induction of HIF-2{alpha} under normoxia (Fig. 3A). Interestingly, rotenone blockade of HIF-2{alpha} induction during hypoxia could be rescued by the addition of dimethyl (R)-(+)-methyl succinate (MR-Succ; 5 mM), a more permeable form of the complex II donor succinate (Fig. 3B). Succinate can donate electrons directly to complex II, bypassing rotenone blockade at complex I, and thereby restore partial electron flow in the mitochondrial ETC.


Figure 3
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  		Fig. 3. Mitochondrial inhibitors block HIF-2{alpha} induction during hypoxia. A: MAH cells were treated with the complex I inhibitor rotenone (Rot; 1 µM), complex III inhibitors myxothiazol (Myx; 1 µM) and antimycin A (Ant A; 1 µg/ml), and the complex IV inhibitor cyanide (CN–; 1 mM) under hypoxia (2% O2, 2 h) and under normoxia in the presence of DFX (100 µM) for 4 h and probed for HIF-2{alpha}. Blocking complex I, III, or IV prevented HIF-2{alpha} induction during hypoxia. DFX induced HIF-2{alpha} under normoxia even in the presence of rotenone, myxothiazol, antimycin A, and cyanide. B: cells were treated with rotenone (1 µM) under normoxic and hypoxic conditions in the presence of dimethyl (R)-(+)-methyl succinate (MR-Succ; 5 mM). MR-Succ partially reversed the inhibitory effect of rotenone on HIF-2{alpha} stabilization during chronic hypoxia.

 
Are reactive oxygen/nitrogen species required for HIF-2{alpha} stabilization in MAH cells? It has been reported that ROS such as H2O2 can induce HIF-{alpha} under normoxic conditions in certain cell lines and that treatment with ROS scavengers can prevent HIF-{alpha} accumulation during hypoxia (2, 15, 20). To test the generality of this idea, we investigated the effects of a variety of ROS (and reactive nitrogen species, RNS) scavengers in MAH cells. The ROS scavengers or antioxidants Trolox (200 µM), N-acetyl-L-cysteine (50 µM), and ascorbic acid (200 µM) failed to prevent HIF-2{alpha} induction during hypoxia (2% O2, 2 h) (Fig. 4, A and B). Although these drugs do not affect HIF-2{alpha} induction, they are still capable of affecting other physiological functions. In voltage-clamp experiments, the application of either N-acetyl-L-cysteine (50 µM) or ascorbic acid (200 µM) to MAH cells inhibited outward K+ (at +30 mV) current by 21.3% and 18.5% (n = 4), respectively, within seconds (S. Brown, M. Zhang, and C. Nurse, unpublished observation). Similarly, the RNS scavenger CPTIO (100 µM) failed to prevent HIF-2{alpha} reduction during hypoxia (Fig. 4A). Moreover, neither the superoxide producer 2,3-dimethoxy-1,4-naphtoquinone (40 µM), H2O2 (40 µM), nor tertbutyl peroxide (40 µM) induced HIF-2{alpha} under normoxia (Fig. 4, B and C). In addition, application of 50 µM and 100 µM of both H2O2 and tertbutyl peroxide also failed to induce HIF-2{alpha} (our unpublished observation). Thus, exogenous ROS failed to induce HIF-2{alpha} in MAH cells under normoxia, even though both H2O2 and tertbutyl peroxide were repeatedly administered in 15-min boluses to avoid evaporative losses from the medium. To verify that effects of both H2O2 and tertbutyl peroxide altered ROS in MAH cells, intracellular ROS levels were measured using the fluorescent probe dichlorodihydroxyfluorescein diacetate. Application of 40 µM H2O2 and tertbutyl peroxide for 20 min increased dichlorofluorescein fluorescence by 2.8- and 3.1-fold, respectively, relative to control (our unpublished observations).


Figure 4
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  		Fig. 4. Effects of reactive oxygen and nitrogen species (ROS/RNS) on HIF-2{alpha} induction. A: effects of ROS/RNS scavengers and ROS donors on HIF-2{alpha} induction. MAH cells were treated with the ROS scavengers Trolox (200 µM) and N-acetyl-L-cysteine (NAC; 50 µM) and the RNS scavenger carboxy-PTIO potassium salt (CPTIO; 100 µM), under normoxia and hypoxia (2% O2) for 2 h. Treatments with RNS/ROS scavengers failed to inhibit HIF-2{alpha} stabilization during hypoxia. B: MAH cells were treated with the superoxide producer 2,3-dimethoxy-1,4-naphtoquinone (DMNQ; 40 µM) and the ROS scavenger or antioxidant ascorbic acid (Asc; 200 µM) under normoxia and hypoxia (2% O2) for 2 h. DMNQ failed to induce HIF-2{alpha} under normoxia, and ascorbic acid failed to block HIF-2{alpha} under hypoxia. C: MAH cells were treated with H2O2 (40 µM) or tertbutyl peroxide (TBPer; 40 µM) in 15-min boluses under normoxia and hypoxia (2% O2). HIF-2{alpha} levels were probed. Both H2O2 and tertbutyl peroxide failed to induce HIF-2{alpha} under normoxia.

 
Effects of mitochondrial inhibitors on O2 consumption. Cellular O2 levels depend on several variables, including metabolic activity, O2 tension, and cell type (10, 16). To learn more about the effects of mitochondrial inhibitors on cellular respiration in MAH cells, we monitored O2 consumption in cells transferred to wells of an oxygen biosensor plate (see MATERIALS AND METHODS) and treated with various blocker concentrations. As indicated in Fig. 5, A–D, the blockers rotenone, myxothiazol, antimycin A, and cyanide caused a dose-dependent decrease in the normalized O2 consumption, plotted as percent respiration. Notably, the concentrations of rotenone (1 µM), myxothiazol (1 µM), antimycin A (1 µg/ml), and cyanide (1 mM) that blocked HIF-2{alpha} induction at 2% O2 (see Fig. 3A) also inhibited O2 consumption by >95% as compared with untreated cells (Fig. 5, A–D). The inhibition of oxygen consumption by rotenone could be partially reversed with the addition of 5 mM MR-Succ (Fig. 5E). Similarly, in mitochondria-deficient {rho}0 MAH cells and mutant cells with selective knockdown of RISP (shRISP) and COX10 (shCOX10), the normalized O2 consumption was decreased by ~80% to >95% (Fig. 5F).


Figure 5
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  		Fig. 5. Oxygen consumption assays on MAH cells in the presence of mitochondrial inhibitors. Cells were treated with the following concentrations: 1 nM, 10 nM, 100 nM, 1 µM, or 5 µM rotenone (A); 1 nM, 10 nM, 100 nM, 1 µM, or 5 µM myxothiazol (B); 1 ng/ml, 10 ng/ml, 100 ng/ml, 1 µg/ml, or 5 µg/ml antimycin A (C); or 1 µM, 10 µM, 100 µM, 1 mM, or 5 mM cyanide (D). E: cells treated with MR-Succ (5 mM) showed partial reversal of the inhibition of oxygen consumption by rotenone (1 µM). *P < 0.05 vs. control; **P < 0.05 vs. rotenone treatment. F: similarly, oxygen consumption of {rho}0 cells and MAH cells expressing shRNA interference molecules for RISP and COX10 is shown. *P < 0.05 vs. control. Data are means ± SE; n = 3.

 
Interaction between O2 concentration and the degree of mitochondrial inhibition on HIF-2{alpha} induction. Our above findings are consistent with the notion that mitochondria are important for HIF-2{alpha} induction in MAH cells during hypoxia. However, because changes in mitochondrial ROS/RNS production did not explain the induction of HIF-2{alpha}, we considered the alternative proposal that it was mediated by oxygen redistribution within the cell (10, 16). To test this idea, we exposed MAH cells to varying levels of hypoxia and varying concentrations of the mitochondrial blockers rotenone, myxothiazol, antimycin A, and sodium cyanide. At moderate-to-severe levels of hypoxia (6% O2, 2 h), there was a moderate HIF-2{alpha} induction that was blocked by low doses of rotenone (10 nM or 100 nM; Fig. 6A). However, at more severe levels of hypoxia (2% O2, 2 h), these lower doses of rotenone were no longer effective. This same trend was observed with myxothiazol, antimycin A, and cyanide (Fig. 6, B–D). These data suggest that the inhibition of HIF-2{alpha} by mitochondrial blockers is dependent on O2 concentration.


Figure 6
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  		Fig. 6. The effects of mitochondrial inhibitors on HIF-2{alpha} levels are dependent on oxygen concentrations. MAH cells were exposed to normoxia, to varying levels of hypoxia (6%, 4%, and 2%), and to various concentrations of mitochondrial inhibitors. A: although low concentrations of rotenone (10 nM and 100 nM) inhibited HIF-2{alpha} induction at moderate-to-severe levels of hypoxia (6% and 4% O2, respectively), they failed to inhibit at more severe levels of hypoxia (2% O2). B: 100 nM myxothiazol inhibited HIF induction at 6% O2 but had no effect at 2% O2. C: 100 ng/ml and 10 ng/ml antimycin A are sufficient to inhibit HIF-2{alpha} at 6% O2 but had no effect at 2% O2 D: 100 µM and 10 µM cyanide blocked induction of HIF-2{alpha} at 6% O2 but failed to inhibit at 4% and 2% O2.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present study, we investigated the mechanisms underlying the induction of the transcription factor HIF-2{alpha} during hypoxia in an O2-sensitive immortalized adrenal chromaffin (MAH) cell line, and particularly the role of mitochondria. As previously reported for a variety of other cell lines (5, 9, 10, 16, 24), we found that functional mitochondria played a critical role in HIF-2{alpha} induction. In particular, genetic perturbation of mitochondrial function using short hairpin RNAi techniques or mitochondria-deficient {rho}0 cells led, in all cases, to an inhibition of HIF-2{alpha} induction during hypoxia. These data were complemented by similar findings based on the more commonly used pharmacological ETC blockers, although the specificity of these blockers has recently become a source of concern.

In some studies, the requirement of a functional mitochondrial electron transposrt chain for HIF-{alpha} induction has been linked to exposures to hypoxia, but not anoxia, and this was attributable to an increase in ROS generation at the Qo site of mitochondrial complex III (2, 5, 15, 20, 21, 29, 31). According to this model, it would be expected that blocking or inhibiting the ETC downstream of the Qo site would have no effect on HIF-{alpha} induction. However, our data using RNAi to knock down the RISP subunit of complex III and the more distal COX10 subunit of complex IV do not support this model. Indeed, in the present study, all mitochondrial inhibitors including both upstream and downstream pharmacological ETC blockers (i.e., rotenone, myxothiazol, antimycin A, and cyanide) inhibited HIF-2{alpha} induction during hypoxia. Moreover, the model itself is contentious because other groups have reported that in several cell lines, inhibition of complex IV (and other mitochondrial complexes) did inhibit HIF-2{alpha} induction (10, 16), similar to our present findings. Of interest is a recent report that HIF-1 can differentially regulate various cytochrome-c oxidase subunits such that COX4-1 is downregulated, whereas COX4-2 is upregulated, during hypoxia (13). Interestingly, their data suggest that cells expressing predominantly COX4-2 versus COX4-1 fail to produce an increase in ROS (e.g., H2O2) during hypoxia. This raises the possibility that the specific expression pattern of subunits of mitochondrial complexes may dictate how different cell types alter their ROS levels under hypoxia. In the present study, we failed to uncover a role for ROS (or RNS) in the induction of HIF-2{alpha} in MAH cells because exogenous application of H2O2 or tertbutyl peroxide (or CPTIO) did not induce HIF-2{alpha} under normoxic conditions. Conversely, HIF-2{alpha} induction during hypoxia could not be blocked by the presence of various ROS/RNS scavengers. Taken together, these data strongly suggest that changes in ROS levels are not critical for HIF-2{alpha} induction during hypoxia in MAH cells.

Our results are consistent with an alternative model where mitochondria act as oxygen "sinks," thereby leading to variations in cellular O2 distribution (34). We tested this theory by treating MAH cells with varying concentrations of mitochondrial inhibitors at different levels of hypoxia. All the inhibitors showed similar results. At moderate-to-severe levels of hypoxia (6% O2) that were sufficient to induce HIF-2{alpha}, low doses of rotenone, myxothiazol, antimycin A, and cyanide were sufficient to inhibit HIF-2{alpha} induction. However, at more severe levels of hypoxia (2%), much higher doses (x100) of the mitochondrial inhibitors were needed to achieve the same inhibition. These data show that the effects of these drugs are O2 dependent. At moderate-to-severe levels of hypoxia, inhibiting the ETC even slightly may "free up" enough oxygen for the PHDs to function and cause degradation of HIF-2{alpha}. At more severe levels of hypoxia, the ETC needs to be inhibited almost completely since oxygen levels are so limited.

In summary, we have shown that functional mitochondria are required for HIF-2{alpha} induction during hypoxia in this immortalized O2-sensitive adrenomedullary chromaffin cell line, but increases in ROS levels do not appear to mediate this response. Furthermore, there does not appear to be a critical site within the ETC for this induction, but rather a requirement for a fully functional ETC. Our conclusion is consistent with a previously proposed model whereby mitochondria generate oxygen gradients within the cytoplasm, greatly limiting the amount of O2 available for the prolyl hydroxylases (PHDs). By inhibiting the mitochondrial ETC (e.g., using {rho}0 cells, pharmacological blockers, or RNAi techniques), this gradient is alleviated or nullified, allowing for proper PHD activity and thus reduced HIF-{alpha} accumulation (Fig. 7, A–C). While our data do not resolve the existing controversies, they provide evidence supporting the theory of mitochondria as oxygen sinks. While increases in ROS levels may be critical for HIF-{alpha} induction in some cells, alternative mechanisms appear to exist in other cell types. Given the current excitement concerning the role of HIF function in cancer, and the development of potential treatment strategies (7, 19), it is important to recognize that multiple mechanisms may be involved.


Figure 7
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  		Fig. 7. Schematic model showing effects of oxygen consumption on HIF-2{alpha} induction. A: under normoxia (21% O2), there is an abundant supply of oxygen for both mitochondrial respiration and the prolyl hydroxylases (PHDs), which, in turn, leads to HIF degradation. B: under hypoxia (2% O2), little oxygen is available and is used primarily by the mitochondria, leaving insufficient amounts for the PHDs to hydroxylate HIF-{alpha}. This allows HIF-{alpha} to accumulate, dimerize with HIF-1β (ARNT), and translocate to the nucleus. C: if mitochondrial function is inhibited during hypoxia, the small amount of available oxygen can be used by the PHDs, leading to the degradation of HIF-{alpha}.

 

   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by operating grants from the Heart and Stroke Foundation (HSF) of Ontario (no. T-5819) and the Canadian Institutes of Health Research (no. MOP-12037) to C. A. Nurse. S. T. Brown was supported by a Focus on Stroke award from HSF of Canada.


   ACKNOWLEDGMENTS
 
We thank Cathy Vollmer for expert technical assistance, Josef Buttigieg for providing {rho}0 cells, Grant McClelland for assistance with the O2 consumption measurements, and Juliet Daniel and Kevin Kelly for assistance in generating retrovirus.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. T. Brown, Dept. of Biology, McMaster Univ., 1280 Main St. West, Hamilton, Ontario, L8S 4K1 Canada (e-mail: brownst{at}mcmaster.ca)

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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