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Carcinogenesis Advance Access originally published online on May 29, 2008
Carcinogenesis 2008 29(8):1528-1537; doi:10.1093/carcin/bgn125
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© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

HIF and reactive oxygen species regulate oxidative phosphorylation in cancer

Eric Hervouet1,6,{dagger}, Alena Cízková2,3,{dagger}, Jocelyne Demont1, Alena Vojtísková2, Petr Pecina2, Nicole L.W. Franssen-van Hal5, Jaap Keijer5, Hélène Simonnet1,7, Robert Ivánek3,4, Stanislav Kmoch3, Catherine Godinot1,* and Josef Houstek2

1 Centre de Génétique Moléculaire et Cellulaire, UMR 5534, Centre National de la Recherche Scientifique, Claude Bernard University of Lyon 1, 43 Boulevard du onze novembre, 69622 Villeurbanne, Cedex, France
2 Institute of Physiology, Academy of Sciences of the Czech Republic, Videnska 1083, 142 20 Praha, Czech Republic
3 Institute of Inherited Metabolic Disorders, Faculty of Medicine, Charles University, Ke Karlovu 2, Prague 12808, Czech Republic
4 Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Videnska 1083, 142 20 Praha, Czech Republic
5 RIKILT—Institute of Food Safety, Wageningen, The Netherlands
6 Present address: Institut National de la Santé et de Recherche Médicale U601, Institute of Biology, 9 quai Moncousu, F-44035 Nantes, France
7 Present address: UMR 5201, Centre National de la Recherche Scientifique, Université Claude Bernard Lyon 1, F-69373 Lyon, France

* To whom correspondence should be addressed. Tel: +33 478364192; Fax: +33 4 72 43 26 85; Email: godinot1{at}yahoo.com


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
 References
 
A decrease in oxidative phosphorylation (OXPHOS) is characteristic of many cancer types and, in particular, of clear cell renal carcinoma (CCRC) deficient in von Hippel–Lindau (vhl) gene. In the absence of functional pVHL, hypoxia-inducible factor (HIF) 1-{alpha} and HIF2-{alpha} subunits are stabilized, which induces the transcription of many genes including those involved in glycolysis and reactive oxygen species (ROS) metabolism. Transfection of these cells with vhl is known to restore HIF-{alpha} subunit degradation and to reduce glycolytic genes transcription. We show that such transfection with vhl of 786-0 CCRC (which are devoid of HIF1-{alpha}) also increased the content of respiratory chain subunits. However, the levels of most transcripts encoding OXPHOS subunits were not modified. Inhibition of HIF2-{alpha} synthesis by RNA interference in pVHL-deficient 786-0 CCRC also restored respiratory chain subunit content and clearly demonstrated a key role of HIF in OXPHOS regulation. In agreement with these observations, stabilization of HIF-{alpha} subunit by CoCl2 decreased respiratory chain subunit levels in CCRC cells expressing pVHL. In addition, HIF stimulated ROS production and mitochondrial manganese superoxide dismutase content. OXPHOS subunit content was also decreased by added H2O2. Interestingly, desferrioxamine (DFO) that also stabilized HIF did not decrease respiratory chain subunit level. While CoCl2 significantly stimulates ROS production, DFO is known to prevent hydroxyl radical production by inhibiting Fenton reactions. This indicates that the HIF-induced decrease in OXPHOS is at least in part mediated by hydroxyl radical production.

Abbreviations: CCRC, clear cell renal carcinoma; DCF, 2',7'-dichlorofluorescein; DFO, desferrioxamine; DMEM, Dulbecco’s modified Eagle’s medium; HIF, hypoxia-inducible factor; Mn-SOD, manganese superoxide dismutase; mRNA, messenger RNA; OXPHOS, oxidative phosphorylation; PCR, polymerase chain reaction; RCC, renal cancer cell; ROS, reactive oxygen species; SOD, superoxide dismutase; siRNA, small interfering RNA; vhl, von Hippel–Lindau


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
 References
 
More than 50 years ago, Warburg observed that cancer cells maintained a high glycolytic rate even in the presence of oxygen. They use glycolysis rather than oxidative phosphorylation (OXPHOS) to produce most of their adenosine triphosphate (1). Whereas the stimulation of glycolysis is explained by an upregulation of glycolytic genes mainly by stabilization of the transcription factor, hypoxia-inducible factor (HIF), the downregulation of OXPHOS is not fully understood.

Previous studies from our laboratory and from others have shown that all mitochondrial respiratory chain activities and subunit contents were lower either in renal tumors exhibiting a deficiency in von Hippel–Lindau (vhl) tumor suppressor gene than in normal adjacent tissue (2,3) or in cultured clear cell renal carcinoma (CCRC) cells (786-0 cells issued from such tumors) before being transfected with vhl (4,5).

HIF is composed of a constitutively expressed HIF1-β subunit and oxygen-sensitive HIF1-{alpha} or HIF2-{alpha} subunits (6). Under aerobic conditions, the HIF-{alpha} subunits are hydroxylated (7) at the level of conserved proline and asparagine residues. After proline hydroxylation, the HIF-{alpha} subunits are ubiquitinated by the vhl tumor suppressor protein, pVHL, which targets them for degradation by the proteasome. Under hypoxia where HIF-{alpha} hydroxylation is reduced or in the absence of pVHL where ubiquitination does not occur, the HIF-{alpha} subunits are stabilized and translocated to the nucleus. They associate with HIF1-β, recruit transcriptional coactivator proteins, such as p300/CPB, and bind to the hypoxia response elements present in >70 known genes, which enhances their transcription. HIF-{alpha} asparagine hydroxylation prevents the binding of transcriptional coactivators (for review, see ref. 8). The prolyl hydroxylases, which contain Fe2+ in their active site, can be inactivated by iron chelators such as desferrioxamine (DFO). Moreover, transition metals such as CoCl2 stabilize HIF by depleting cellular ascorbate, essential cofactor of prolyl hydroxylases (9). As early as 1996, Semenza et al. (10) showed that activation of glycolytic genes by HIF1-{alpha} increased conversion of glucose to pyruvate and lactate. In addition, activation of the HIF-targeted kinase, pyruvate dehydrogenase kinase inhibiting pyruvate dehydrogenase, suggested that respiration was decreased by substrate limitation (11). More recently, Fukuda et al. (12) suggested that HIF-1{alpha} could upregulate the COX4-2 isoform (13) and the LON protease required for COX4-1 isoform degradation. The ensuing COX4-1/COX4-2 isoform switch could increase the cytochrome c oxidase (COX) activity at low oxygen concentration because the COX4-2 isoform imposes higher turnover than the COX4-1 isoform. The switch could then improve electron flux through the respiratory chain.

Simultaneously to the restoration of respiratory chain protein complexes observed after vhl transfection in 786-0 cells, their capacity to rely on OXPHOS for their growth was also increased (4). However, the induction of COX4-2 could not be observed in these cells (14). This result that appears in contradiction with that observed by Fukuda et al. (12) can probably be explained by the fact that these cells express HIF2-{alpha} but not HIF1-{alpha} (15). This would indicate that COX4-2 can only be induced by HIF1-{alpha}. Therefore, additional mechanisms parallel to the COX4 subunit isoform switch should be involved in the downregulation of respiratory chain complexes in these cancer cells.

In the present paper, we have studied whether inhibition of HIF synthesis by RNA interference could restore the level of respiratory chain subunits in the absence of pVHL and whether stabilization of HIF by compounds such as DFO or CoCl2 that prevents HIF hydroxylation would decrease their content in the presence of pVHL. The COX4 subunit was initially chosen as the preferred target. Then, since all mitochondrial respiratory chain activities and their subunit contents were decreased in these cancer cells, we also studied two complex III proteins, the Core 2 subunit and the 13.4 kDa subunit that were markedly modified by the presence or absence of pVHL (4,5). Complex III is particularly relevant to this study because it is known as one of the primary site of mitochondrial reactive oxygen species (ROS) production during hypoxia (16) and participates in the production of superoxide anions (17). Indeed, this study will show that an enzyme involved in ROS production such as the mitochondrial manganese superoxide dismutase (Mn-SOD) is markedly induced in vhl-deficient cells and that increases in ROS production are correlated with the downregulation of respiratory chain subunits.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Supplementary material
 Funding
 References
 
Cell culture
pVHL-deficient parental 786-0 cells that had been stably transfected either with a vector expressing wild-type vhl (VHL+ cells, clones WT8) or with a void vector (VHL– cells, clone PRC3) (18) were kindly provided by Dr Kaelin. Cells were routinely grown as described in (4) and used at 70–80% confluence. Cells were counted in the presence of 0.04% trypan blue.

Comparative microarray analysis of gene expression after transfection of 786-0 cells with a vector expressing pVHL or with a void vector
Total RNA was extracted using the TRIZOL (Invitrogen, Carlsbad, CA), quantified and purity checked by NanoDrop spectrophotometer (NanoDrop Technologies, Rockland, DE) and 1% tris-Borate-EDTA/agarose gels. Quality control was performed on Agilent 2100 bioanalyser—RNA Lab-On-a-Chip (Agilent Technologies, Palo Alto, CA). Total RNA (50 µg) was directly labeled by incorporation of either Cy3-dCTP or Cy5-dCTP during reverse transcription. Complementary DNA was purified using the polymerase chain reaction (PCR) purification protocol (QIAquick PCR purification kit, QIAgen, Düsseldorf, Germany). For each cell line, three biological replicates were analyzed in duplicate against common reference (RNA pool aliquots from all the experiments) using an oligonucleotide microarray containing Human 10K Oligo Set (MWG, Raleigh, NC) spotted on Ultra GAPSTM Coated Slides (Corning, NY). The microarray preparation, hybridization conditions and data acquisition were performed as described previously (19,20). Image analysis of the resulting TIFF files was done and expression data were obtained using Gene PixPro software (Axon Instruments, Union City, CA). Data analysis was performed in R statistic environment (http://www.r-project.org/) using linear models for microarray data with Limma package (21) that is part of Bioconductor project (http://www.bioconductor.org/). Normalization of gene expression data was done by Loess and G-quantile function, and background was corrected by multiple testing corrections using the false discovery rate method of Benjamini et al. (22).

Reverse transcription–PCR
Total RNA was extracted with TrizolTM reagent (InvitrogenTM). Complementary DNA was prepared as before and used for semiquantitative PCR or quantitative PCR [Light cycler instrumentTM, in the presence of SYBR-GreenTM (Roche Diagnostics, Basel, Switzerland)] using previously described primers (4,14). Genomic DNA of 786-0 VHL+ cells, used as positive control of primers, was extracted with the ‘GeneEluteTM mammalian genomic DNA miniprep kit’ (Sigma, St Louis) as recommended by the manufacturer.

RNA interference
The small interfering RNA (siRNA) duplex oligonucleotides designed by Sowter et al. (15) to target HIF2-{alpha} were used. They are targeted to nucleotides 1260–1280 of the HIF2-{alpha} messenger RNA (mRNA) (NM001430) and have the following sequences: sense 5'-CAGCAUCUUUGAUAGCAGUdTdT-3' and antisense 5'-ACUGCUAUCAAAGAUGCUGdTdT-3'.

The duplexes were prepared by mixing 14 µl of LipofectamineTM (Invitrogen) with 80 nM oligonucleotides in 700 µl Dulbecco’s modified Eagle’s medium (DMEM) for 30 min at room temperature. 786-0 cells were grown in DMEM supplemented with glutamine and 10% fetal calf serum (Invitrogen), in 25 cm2 dishes up to 60% confluence before transfection. The cells were rinsed with DMEM to remove any residual serum before addition of 2 ml DMEM plus the oligonucleotide duplex (final oligonucleotide concentrations: 20 nM) in serum-free conditions. Four hours after transfection, 10% fetal calf serum was added. Cells were studied from 24 to 72 h after transfection.

ROS production
Intracellular ROS levels were estimated with the fluorescent dye 2',7'-dichlorodihydrofluorescein diacetate (Molecular Probes, Eugene, OR) which is a non-polar, non-fluorescent compound that is converted into a polar non-fluorescent derivative (dichlorofluorescein diacetate) by cellular esterases. Dichlorofluorescein diacetate is membrane permeable and is rapidly oxidized to the highly fluorescent 2',7'-dichlorofluorescein (DCF) in the presence of intracellular ROS. Cells cultured in 96-well dishes until 70–80% confluence were incubated in saline buffer (135 mM NaCl, 5 mM KCl, 0.4 mM KH2PO4, 1 mM MgSO4, 20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, pH 7.4, 5.55 mM glucose and 1 mM CaCl2) with 1 µM 5-(and 6)-chloromethyl-2'7'-dichlorodihydrofluorescein diacetate acetyl ester. ROS production was estimated by measuring fluorescence immediately after probe addition and 2 h later (fluorescence plate reader, VictorTM, PerkinElmer, Massachusetts) DCF: {lambda}exc = 485 nm, {lambda}em = 535 nm. Cell number was then estimated in each well by crystal violet staining (23). The fluorescence intensity was normalized to the cell number.

Western blot analysis
Proteins extracted from cell pellets were homogenized in lysis buffer, quantified using the Bradford reagent, resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis in 6 or 12.5% acrylamide/bisacrylamide gel and transferred to a BA83 nitrocellulose membrane (Schleicher and SchüellTM, New Hampshire), as described (4). Staining the gels and membranes either with Coomassie blue or Ponceau Red checked equal loading and protein transfer efficiency. Membranes were blocked with 5% skim milk in NaCl–phosphate buffer and then incubated overnight at 4°C with different primary antibodies, washed five times with buffer A (NaCl–phosphate buffer containing 0.1% Tween 20), incubated for 1 h with horseradish peroxidase-conjugated anti-rabbit IgG (1/10 000) for polyclonal antibodies or anti-mouse IgG (1/5000) (Bio-RadTM, Marnes-la-Coquette, France) for monoclonal antibodies and washed with buffer A. Primary antibodies directed against the OXPHOS proteins were used as described previously (4). Polyclonal anti-HIF2-{alpha} (1/1000) (AbcamTM, Paris, France), anti-actin (1/1000) (Santa Cruz technologiesTM, Santa Cruz), anti-NOX4 (1/1500) (generous gift of B.Goldstein) (24), anti-aconitase (1/2000) (25) and anti-IRP2 (1/1000) (generous gifts of Dr E.Leibold) (26) were used. For analysis of the content of superoxide dismutases (SODs), whole-cell extracts or samples of mitochondria isolated from cultured cells using hypotonic shock were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis in 10% gel, blotted to polyvinylidene difluoride membrane (0.45 µm pores, Millipore, Billerica) and incubated with polyclonal anti-SOD1 (cytosolic Cu-Zn-SOD) (Calbiochem, Gibbstown, no. 574597) and polyclonal anti-SOD2 (mitochondrial Mn-SOD) (Calbiochem, no. 574596). Amounts of proteins revealed by western blot analysis were estimated using either the Molecular AnalystTM or AIDA software.

Iron titration
Hydroxyl radical (•OH) that is mainly responsible for the oxidation of macromolecules is formed by the Haber–Weiss reaction as well as by iron-catalyzed Fenton reactions. Therefore, the ability of cells to concentrate iron can enhance oxidative damages. Intracellular level of Fe2+ was estimated according to Arrigo et al. (23).

Statistical analysis
When indicated, data are given as means ± SDs with n indicating the number of experiments. One-way analysis of variance and t-tests were applied for statistical analysis, as appropriate, using Prism SoftwareTM. Values were considered significant when *P < 0.05, **P < 0.01 or ***P < 0.001.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Supplementary material
 Funding
 References
 
Inactivation of HIF2-{alpha} by siRNA reversed respiratory chain deficiency in VHL-deficient cells
A decrease in OXPHOS activities correlating with a decrease in respiratory chain subunit contents had been observed previously in renal tumors and in cells lacking functional pVHL (2,4). These cells had to rely on glycolysis to sustain their growth. To know whether this OXPHOS decrease in VHL– cells was directly mediated by accumulation of HIF2-{alpha} subunits, siRNA targeted to HIF2-{alpha} was introduced into pVHL-deficient 786-0 cells that had been stably transfected either with a void vector, VHL– cells (786-0 PRC3) or with a vector expressing functional pVHL, VHL+ cells (786-0 WT8). The 786-0 cells are well suited to answer this question since they are devoid of HIF1-{alpha} and the used siRNA treatment prevents the expression of HIF2-{alpha} in these cells (15). In agreement with these authors, no cell toxicity was noted after transfection of the siRNA or Lipofectamine alone. Figure 1A shows that, as expected, the HIF2-{alpha} transcript amount was strongly decreased in the presence of siRNA specific for HIF2-{alpha} in the two cell types. The expression of mRNA coding the constitutively expressed L32 ribosomal protein, used as a control, appeared at the same cycle N in all the different conditions, indicating that each sample contained similar mRNA amounts. The HIF2-{alpha} signal appeared about six cycles later when comparing cells treated in the presence or absence of siRNA-targeting HIF2-{alpha} for VHL+ cells, proving that siRNA had been efficient to decrease HIF2-{alpha}. Moreover, the transfection of the siRNA-targeting HIF2-{alpha} almost completely abolished HIF2-{alpha} protein content, either in VHL– cells or in VHL+ cells treated with CoCl2 to prevent the pVHL-induced degradation of HIF2-{alpha} (Figure 1B).


Figure 1
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  		Fig. 1. Increase of respiratory chain subunit contents in CCRC cells after inhibition of HIF2-{alpha} mRNA expression by treatment with siRNA targeted to HIF2-{alpha}. (A) VHL-deficient 786-0 cells transfected with a void vector (786-0 PRC3: VHL–) or with a vector expressing HIF2-{alpha} (786-0 WT8: VHL+) were treated for 24 h with siRNA targeted to HIF2-{alpha} and the HIF2-{alpha} mRNA expression was analyzed by semiquantitative reverse transcription–PCR. The amplicon amounts observed after N or N + 6 PCR cycles were analyzed by agarose gel electrophoresis and the HIF2-{alpha} mRNA contents were compared with control (L32 ribosomal protein). (B) Decrease in HIF2-{alpha} protein content 48 h after siRNA treatment: western blot analysis using an anti-HIF2-{alpha} antibody was performed with proteins extracted from 786-0 WT8 (VHL+) or 786-0 PRC3 (VHL–) cells treated or not with siRNA directed to HIF2-{alpha} for 24 or 48 h. Treatment with 250 µM CoCl2 was performed for 4 h. The 24 h treatment with the anti-HIF2-{alpha} siRNA was sufficient to prevent the CoCl2-induced HIF2-{alpha} expression. HIF2-{alpha} expression was also prevented 48 h (or 72 h, data not shown) after anti-HIF2-{alpha} siRNA transfection in VHL– (786-0 PRC3) cells. Loading control: western blot stained with Ponceau red indicating similar protein contents in the different samples. (C–E) Restoration of respiratory chain subunit contents by the anti-HIF2-{alpha} siRNA treatment: representative western blot analysis of OXPHOS subunit contents using COX4, Core 2 and ATPase β-subunit (5G11) antibodies (C) performed with 786-0 PRC3 (VHL–) and 786-0 WT8 (VHL+) treated or not for 72 h with siRNA directed to HIF2-{alpha}. Anti-ATPase β-subunit antibody was used as a loading control. Data (D and E) are given as the mean ± SD of five different experiments.

 
Figure 1C shows that, in the absence of siRNA, the amounts of Core 2 (complex III) and COX4 (complex IV) subunits were much lower in VHL– than in VHL+ cells while the ATPase β-subunit content did not significantly change. These data are in agreement with those described previously (4). Transfection with the siRNA-targeting HIF2-{alpha} increased the contents of these respiratory chain subunits in VHL– cells. Therefore, HIF2-{alpha} decreased respiratory chain subunit amounts and HIF2-{alpha} acted downstream pVHL in the regulation of respiratory chain subunit biogenesis. Within 72 h of RNA interference treatment, the amounts of cytochrome c oxidase COX4 subunit (Figure 1D) and complex III Core 2 protein (Figure 1E) had approximately doubled. Similar data were observed with the 13.4 kDa complex III subunit (data not shown). Transfection with the siRNA did not change the amount of these respiratory chain subunits in VHL+ cells in which the HIF2-{alpha} subunit was absent, demonstrating the essential and specific role of HIF2-{alpha} to downregulate these respiratory chain subunits.

It should be reminded that the above experiments have been performed with 786-0 cells that express HIF2-{alpha} but not HIF1-{alpha} (15). In these cells, the COX4 subunit that was titrated with the anti-COX4 antibody must be the COX4-1 isoform. Indeed, we could have never detected the transcript encoding the COX4-2 subunit isoform by reverse transcription–PCR, even in the presence of CoCl2 that stabilizes HIF-2{alpha} in VHL+ cells. This cannot be due to a technical PCR problem since the COX4-2 primers designed within a single exon could efficiently amplify this gene when genomic DNA extracted from VHL– or VHL+ cells was used as template (14).

Differential effects of HIF2-{alpha} stabilization by CoCl2 or DFO on respiratory chain subunit content
To confirm the downregulation of respiratory chain proteins by HIF2-{alpha}, we then studied whether drugs known to stabilize HIF2-{alpha} such as CoCl2 and DFO could also decrease respiratory chain subunit level in cells expressing pVHL. Total proteins were extracted from VHL+ cells after incubation for 8 h with 250 µM DFO or CoCl2. HIF2-{alpha} and COX4-1 contents were analyzed by western blot (Figure 2A and B). As expected, a high signal was detectable with anti-HIF2-{alpha} antibody after 8 h of incubation with CoCl2 or DFO while untreated VHL+ cells did not significantly express HIF2-{alpha}. As shown previously (14), the COX4-1 amount was decreased by CoCl2 (Figure 2A and B). However, no significant difference in COX4-1 amount could be detected between cells treated or not with DFO. The contents of Core 2 and 13.4 kDa complex III subunits were also decreased by CoCl2, but were both not modified by the presence of DFO (data not shown). Hence, although the HIF2-{alpha} content was highly induced in the presence of DFO, DFO did not cause the HIF-induced decrease of respiratory chain subunit levels.


Figure 2
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  		Fig. 2. Influence of VHL presence or HIF-2{alpha} stabilization on COX4-1 level and ROS production. (A and B) Effects of CoCl2- or DFO-stabilizing HIF2-{alpha} on COX4-1 subunit content. The COX4-1 subunit content was estimated by western blot, using VHL+ (786-0 WT8) cells untreated or treated with 250 µM CoCl2 or DFO to stabilize HIF2-{alpha}. Twenty micrograms of proteins were used in each lane. Representative blot is shown in (A). The graph (B) shows the mean ± SD of three experiments. (C) Increased expression of Mn-SOD and in 786-0 cells devoid of pVHL: the content of SODs (Mn-SOD and Cu-Zn-SOD) was quantified by western blot. For analysis, 15 µg of protein from cell extracts or 5 µg of mitochondrial protein were loaded. (D) Effects of HIF expression on ROS production, as measured by DCF fluorescence. The DCF fluorescence intensity was measured as described in the experimental section. Data are given as means ± SDs of three to five experiments, each one being made in triplicates. CoCl2 was incubated for 8 h at a concentration of 250 µM. (E and F) Effects of HIF expression on aconitase activity and contents. Aconitase activity was measured by the method of Drapier et al. (27) in 786-0 PRC3 cells (VHL–) or 786-0 WT8 cells (VHL+) untreated or treated for 8 h with 250 µM CoCl2. To lyse the cells, 2–5 x 106 cells homogenized in 300 µl of 50 mM Tris–HCl, pH 7.4, containing 0.625 mM MgCl2 were sonicated 10 times for 1 s (Vibra Cell 72405 sonicator) and centrifuged at 2000g for 6 min. The aconitase amounts were determined by western blot analysis quantified by densitometry. The different cells and treatments were compared with the untreated 786-0 WT8 cells taken as 100%. Data are given as means ± SDs of three to nine experiments.

 
Effect of pVHL deficiency on the transcriptome of 786-0 cells: microarray analysis of transcripts expressed in 786-0 cells containing either an active or non-functional pVHL
In order to better understand the mechanisms involved in the regulation of respiratory chain subunit amounts by the pVHL/HIF system in 786-0 cells, we have studied the influence of pVHL on mRNA expression by a large-scale microarray analysis. The MWG microarray that was used comprised a set of 10 162 genes. About 6000 gave a valid signal. At a P value <0.0001, 27 genes were upregulated and 77 were downregulated (Table I) when comparing VHL+ to parental VHL–. Supplementary Tables 1 and 2 (available at Carcinogenesis Online) report differences observed with a P value <0.05. Expected differences in the expression of vhl or well-known HIF-induced genes such as vegf or cyclin D1, for example, confirmed the validity of the data.


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  		Table I. Modification of gene expression in 786-0 WT8 cells (VHL+) compared with 786-0 parental cells (VHL–) (P < 10–4)

 
Among differentially expressed genes, none concerned OXPHOS subunits and OXPHOS-specific assembly factors and differences in the expression of only very few genes known to be directly involved in mitochondrial metabolism could be put forward. There was no significant change in promitochondrial regulatory genes, such as PGC1{alpha}, NRF1 and TFAM, and only a slight decrease of p53 in VHL– cells. Therefore, these data confirmed that decreased content of respiratory chain proteins did not probably result from lower expression of their genes but probably from changes in posttranscriptional regulation. Among the overexpressed genes, the mitochondrial Mn-SOD was 7.2-fold more expressed in VHL– than in VHL+ cells. Another gene encoding lysyl oxidase, LOX4 known as a HIF target (28), was also increased by a factor of 12. The importance of these changes observed in Mn-SOD and lysyl oxidase, two enzymes producing H2O2, underscored the role that ROS might play in the regulation of the metabolism of these cells and ROS metabolism was therefore further studied.

Influence of HIF on SODs, aconitase, NOX4 and iron-regulatory proteins
On the basis of these microarray data, the expression of mitochondrial Mn-SOD (Type 2) and cytosolic Cu-Zn-SOD (Type 1) was analyzed in 786-0 cells. Figure 2C shows that, in agreement with the microarray data, the mitochondrial Mn-SOD was markedly stimulated when VHL– were compared with VHL+ cells. There was 6-fold specific content of Mn-SOD in cell lysates and 4-fold in isolated mitochondria of VHL+ cells. On the contrary, no significant change could be observed in the level of the cytosolic Cu-Zn-SOD that also did not appear among the differentially expressed genes in the microarray analysis. Therefore, the cells devoid of pVHL have a particularly efficient tool to transform superoxide anions produced by the mitochondria into H2O2.

2',7'-Dichlorodihydrofluorescein diacetate represents a well-established fluorescence probe to monitor mitochondrial ROS production in intact cells (29). Although DCFDH is not a good quantitative probe for measuring H2O2, it detects very well several other oxygen radicals and can be used as a qualitative marker of the overall oxidative stress (30). Figure 2D shows that DCF fluorescence was significantly higher in VHL– cells devoid of active pVHL (hence, expressing HIF2-{alpha}) than in VHL+ cells transfected with functional vhl (hence, inducing HIF2-{alpha} degradation). CoCl2 treatment further increased DCF fluorescence. This technique could not be used in the presence of DFO that complexes iron since oxidation of DCFH to DCF is known to occur slowly if at all in the absence of ferrous ion (30). Therefore, DCF fluorescence is not a valid test for measuring ROS in the presence of DFO.

Aconitase is an iron–sulfur protein that is an essential mitochondrial enzyme and is highly susceptible to oxidative damage. Loss of its activity is frequently used as an indicator of ROS generation. Under conditions of increased hydrogen peroxide concentration, the loss of activity becomes irreversible as a result of Fenton reaction (31). Such conditions occur in VHL– cells due to Mn-SOD upregulation. Indeed, the aconitase activity was significantly lower in VHL– than in VHL+ cells (Figure 2E). However, the aconitase content was only slightly lower in VHL– than in VHL+ cells. This decrease was, however, below the limit of significance (Figure 2F). Treating the VHL+ cells with CoCl2 for 8 h also decreased the aconitase activity (Figure 2E) and did not modify the amount of aconitase (Figure 2F). Importantly, these findings indicate inevitable increase of hydroxyl radical production in VHL– cells.

No significant difference could be put forward for NOX4, a nicotinamide adenne dinucleotide oxidase (data not shown) that is known to be involved in hydrogen peroxide metabolism (24). In parallel, as an increased level of iron could be responsible for increased ROS production, we compared iron contents and IRP1 and IRP2 (data not shown) that are iron-regulatory proteins important in iron transport in the cell (25,26). No significant differences could be detected between VHL– and VHL+ cells (data not shown).

Effects of H2O2 on OXPHOS subunit contents and cell viability
To test the direct effects of H2O2 on OXPHOS subunit contents, VHL+ cells were incubated with increasing concentrations of H2O2 for 8 h. Figure 3A and B shows that COX4-1 and Core 2 protein amounts were both decreased after exposition to H2O2. The decrease observed for COX4-1 was more pronounced than for Core 2, which can be at least partly explained by the slower turnover of Core 2 and 13.4 kDa subunits than that of COX4 (14). Importantly, Figure 4A shows that incubation of the cells for 24 h with increasing amounts of H2O2 was more toxic for cells expressing HIF (VHL–) and susceptible to the production of higher levels of hydroxyl radicals (Figure 2) than for cells in which HIF degradation was induced by pVHL: at 50 µM H2O2, the cell number had been decreased by >50% in VHL– cells while about 75% of the VHL+ cells were still alive. DFO was also more toxic for VHL– cells than for VHL+ cells. However, the presence of 50 or 100 µM DFO, an agent inhibiting Fenton reaction, diminished the H2O2-induced cell toxicity, especially in the case of VHL– cells (Figure 4A). Indeed, the DFO-induced protective effect against H2O2 was not very efficient in VHL+ cells. We have seen above (Figure 2B) that DFO was also able to partly protect VHL– cells against HIF2-{alpha}-induced COX4-1 subunit degradation.


Figure 3
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  		Fig. 3. (A and B) Effect of H2O2 on COX4 and Core 2 contents: VHL+ cells were treated for 8 h with increasing H2O2 concentrations. Total proteins (10 or 20 µg) were analyzed by western blot using anti-COX4 or anti-Core 2 antibodies. (B) Estimation of protein amounts remaining after 8 h of treatment with the indicated H2O2 concentrations. The intensity of the assays tested with the COX4 or Core 2 antibodies in the absence of inhibitor was arbitrary taken as 100% and the intensities observed in the presence of H2O2 was compared with that of this reference. Data are given as mean ± SD of four experiments. Asterisks (*P < 0.05; **P < 0.01) indicate significant differences observed between assays made in the presence of the indicated H2O2 concentrations and the assays made in the absence of H2O2.

 


Figure 4
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  		Fig. 4. Combined effects of H2O2 and DFO on cell growth and COX4-1 contents. (A) The cells were plated in 96-well plates at a density of 104 cells per well and incubated on the following day for 24 h in the presence of H2O2 and/or DFO at the indicated concentrations. Cell number was estimated by crystal violet staining. Each point represents an average of six determinations. Top left: effects of increasing H2O2 concentrations on the viability of 786-0 cells transfected with a void vector (VHL–) or with a vector expressing functional pVHL (VHL+). Top right: effects of increasing DFO concentrations on the viability of 786-0 cells transfected with a void vector (VHL–) or with a vector expressing functional pVHL (VHL+). Influence of DFO at concentrations of 50 or 100 µM on H2O2-induced cell toxicity in VHL– (bottom left) or VHL+ (bottom right) cells. (B–D) Combined effects of H2O2 and DFO on COX4-1 subunit contents in VHL– or VHL+ cells. The cells (106 cells per 25 cm2 flask) were treated for 24 h in the presence or absence of 100 µM H2O2 and/or 100 µM DFO. For analysis, 20 µg of proteins were loaded in each lane. The COX4-1 content was quantified by western blot. (B) Loading control stained with Ponceau red showing similar protein contents in the different samples. (C) Western blot obtained using anti-COX4 antibody. In each experiment, the sample corresponding to VHL+ cells in the absence of H2O2 and DFO (sample 2) was arbitrarily fixed at 100. The spot intensities were then calculated by comparison with this arbitrary value. The mean ± SD of three experiments is reported on graph (D). Bars with gray and white background represent VHL– and VHL+ cells, respectively. Asterisks (*P < 0.05; **P < 0.01) indicate significant differences observed between treatments when comparing assays linked by brackets.

 
Figure 4C and D shows that, as expected, the VHL+ cells always contain more COX4-1 than the VHL– cells incubated under the same conditions (compare lanes 1 to 2, 3 to 4, 5 to 7 or 6 to 8). In addition, when COX4-1 amount is decreased by H2O2 treatment (compare lanes 1 to 3 or 2 to 4), the presence of DFO restored the normal COX4-1 level (compare lanes 3 to 6 or 4 to 8).


   Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Supplementary material
 Funding
 References
 
Previous studies from our laboratory had shown that, in agreement with observations made on several cancer types (32,33), including the early observations of Warburg (1), OXPHOS is downregulated in renal cancer cells (RCCs) (2). In CCRCs, vhl-encoding pVHL is one of the main genes involved in tumorigenesis. When vhl is transfected in vhl-deficient CCRCs, the cells loose their capacity to develop tumors in nude mice (34) and their OXPHOS subunit contents are restored (4). In this paper, the use of siRNA targeted to HIF2-{alpha} in cells devoid of HIF1-{alpha} suggested that the presence of HIF was primarily responsible for the downregulation of respiratory chain subunit biogenesis. The amount of OXPHOS subunits was inversely correlated to the expression of HIF-{alpha} independently of pVHL. Indeed, in the VHL– cells, the inhibition of HIF2-{alpha} expression by siRNA increased the content of OXPHOS subunit, as shown for complex III (Core 2; 13.4 kDa subunits) or complex IV (COX4-1 subunit). The effect was specific since siRNA targeted to HIF2-{alpha} modified neither cell viability nor OXPHOS subunit content in control VHL+ cells.

Fukuda et al. (12) recently confirmed that transfection of vhl in another line of vhl-deficient RCCs, RCC4, could also restore COX deficiency. They proposed that HIF1 could induce COX4-2 subunit after binding to a hypoxia response element present in the COX4-2 gene 5' untranslated region; on the contrary, the COX4-1 isoform would be degraded probably because of HIF1-induced increased expression of the LON mitochondrial matrix protease. However, this upregulation of COX4-2 is not relevant in 786-0 cells used here since no COX4-2 expression could be detected in these cells. Contrarily to RCC4 cells, the 786-0 cells do not contain HIF1-{alpha} but only HIF2-{alpha} whereas RCC4 cells express both isoforms. The differential COX4-2 expression adds to the already observed differences between these two factors. While some of their effects may be inherent to both, many seem to be quite specific or even antagonizing, such as their differential effect on c-Myc transcriptional activity (35). The absence of COX4-2 transcript also shows that the putative induction of COX4-2 transcription regulated by a novel oxygen-responsive element described by Hüttemann et al. (36) did not occur in these cells, which could be expected since, in culture, the cells were not submitted to hypoxia and since, in vivo, COX4-2 transcription was seen in lung and liver, but not in brain, heart and kidney (13).

As expected from the above RNA interference experiments, HIF stabilization by CoCl2 in VHL+ cells also decreased respiratory chain subunit contents. However, since CoCl2 inhibits the mitochondrial intermediate peptidase, which prevents pre-COX4 cleavage and hence cytochrome c oxidase assembly (14), the relationship between CoCl2 effects on HIF stabilization and on respiratory chain subunit degradation could not be established without ambiguity. In addition, DFO induced HIF2-{alpha} overexpression but, unexpectedly, did not decrease respiratory chain subunits content, suggesting that the presence of HIF2-{alpha} was not sufficient in the presence of DFO.

Since HIF is a transcription factor involved in the regulation of a large panel of genes, we looked for differences in gene expression that could explain the HIF-induced OXPHOS downregulation by using large-scale microarray analysis. Mitochondrial biogenesis is regulated at the level of transcription by expression of several nuclear respiratory factors such as NRF1 or NRF2, which is stimulated by PPRA{gamma} coactivator-1 or PGC-1 related coactivator (see ref. 37 for review). The binding of NRF1 or NRF2 to promoters or enhancers of numerous OXPHOS subunit genes stimulates their transcription. Although a decrease in respiratory chain proteins is well documented in renal cancer (2,3,15) as well as in many other cancer types (32,33), a decrease in the expression of these transcription factors has not been reported, neither by large-scale microarray nor by serial analysis of gene expression (SAGE) analysis comparing tumoral to normal adjacent tissue (38) or pVHL-deficient RCCs transfected or not with wild-type vhl (39). Our study confirmed that the expression of these transcription factors or transcripts coding for OXPHOS complex subunits did not correspond to the major differences observed in protein contents when comparing VHL– to VHL+ cells. Microarray data (Table I and supplementary Tables 1 and 2, available at Carcinogenesis Online) did not reveal significant differences between VHL– and VHL+ cells for LON protease or COX4-2 gene expression. However, our study shows prominent changes in the expression of genes involved in ROS metabolism such as the mitochondrial Mn-SOD or the LOX4 lysyl oxidase. Importantly, this was paralleled by protein contents of Mn-SOD (Figure 2D), which is located in the matrix where it dismutates superoxide anions into hydrogen peroxide.

Several reports have implied ROS production in HIF-{alpha} stabilization in various types of cancer cells (see refs 16,40 for review). According to Schroedl et al. (41), an increase in ROS production during hypoxia can control HIF-{alpha} activation. Furthermore, Okamoto et al. (42) observed a higher content of ROS-modified nucleotides and proteins in human renal cell carcinoma, showing that RCC constitutively elaborates more ROS than is produced by the non-tumorous parts of kidneys. Accordingly, our data also showed that the absence of pVHL, which results in HIF stabilization, is associated with a ROS increase inducing a higher DCF fluorescence (Figure 2D) and a decreased aconitase activity (Figure 2E). In respiratory chain, the ROS production originates from one electron leak reacting with oxygen to give primary oxygen radicals, superoxide anions (43). The increased ROS production by mitochondrial respiratory chain is often compensated by upregulation of intramitochondrial SOD (Mn-SOD) and other scavenging mechanisms. The increase in Mn-SOD, observed by proteomic analysis of CCRC compared with normal adjacent tissue (44,45) or in VHL– cells in our study (Figure 2C), would then cause conversion of intramitochondrially generated superoxide anions in these cells thus increasing the level of hydrogen peroxide. The Mn-SOD increase might be a mechanism that the cell developed to survive. Indeed, the VHL– cells are more susceptible to H2O2 toxicity than VHL+ cells (Figure 4A). Since an increase in H2O2 destroyed respiratory chain subunits (Figure 3A and B), there has to be an autoregulation between levels of HIF, ROS and OXPHOS subunit content. ROS production stabilized HIF but decreased OXPHOS subunit contents. This can in turn decrease ROS production through complex I and III downregulation (46).

The mitochondrial Mn-SOD increase we observed in VHL– cells (Figure 2D) leads to enhanced conversion of superoxide radicals to molecular oxygen and H2O2. H2O2 being membrane permeable can then be carried out toward other cell compartments and be converted to water in the presence of catalase or peroxidases present in these compartments. However, the toxicity vastly increases when H2O2 is transformed into hydroxyl radicals by Fenton reactions, which exactly seems to occur in VHL– cells. This hypothesis is strongly supported by experiments with DFO, which prevents Fenton reactions by complexing iron (30) and hence inhibits the production of hydroxyl radicals. This explains why DFO treatment did not decrease respiratory chain subunit amounts in VHL+ cells, whereas the other HIF-stabilizing compound CoCl2 decreased them similarly to what was observed in VHL– cells. This shows that ROS enhanced by HIF stabilization are important factors responsible for HIF-induced respiratory chain downregulation. The fact that DFO protects the VHL– cells against H2O2-induced degradation of COX4 subunit (Figure 4C and D) suggests that the ROS species most important in OXPHOS subunit degradation are the hydroxyl radicals produced by the Fenton reactions. The scheme shown in Figure 5 summarizes the various ways discussed in this paper by which HIF and ROS modify energy metabolism.


Figure 5
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  		Fig. 5. Scheme summarizing the main effects of HIF and various ROS components on cell bioenergetics. Thick plain arrows indicate genes targeted by HIF; thick dotted lines with arrows show targets for ROS; ‘{intcal}’ means direct inhibition. See Introduction for details on HIF/VHL mechanisms and Discussion for ROS involvement.

 
While the mechanisms by which ROS modulate HIF stability and regulation of target gene expression have been thoroughly investigated in previous reports, this paper shows that the ROS increase induced by HIF stabilization appears to be essential to downregulate the level of respiratory chain subunits. However, the reason why HIF increases ROS production remains hypothetical. ROS production might be a consequence of hindered electron transfer through respiratory chain complexes. The alteration of respiratory chain complexes involves changes in electron flux. Decreasing COX activity decreases the rate of oxygen utilization and leaves more oxygen available for superoxide anions production through complex III or complex I. That a decrease in complex III can increase ROS production is more puzzling. This paradox has already been observed in mitochondrial diseases involving cytochrome b mutations and showing increase in ROS production. In fact, we cannot also exclude that the downregulation was connected with some qualitative changes in the complex thus promoting electron leak and ROS production.

Although ROS are important in stabilization of HIF that maintains the transcription of genes involved in tumor development, a ROS excess is toxic for the cancer cell and ROS level should be precisely regulated. Similarly, Burdon (47) had shown that a slight H2O2 increase conferred a proliferative advantage to cancer cells while higher H2O2 concentrations were toxic. Since one of the main sources of ROS is provided by the respiratory chain complex I and complex III (46,48,49), the decrease in OXPHOS may be a crucial step in the development of tumor cells to avoid excessive ROS toxicity. Direct ROS increase after H2O2 addition to VHL– cells also had a deadly effect to the cells. In conclusion, tumor cells must finely tune the level of ROS upregulating them to stabilize HIF and preventing their excess to avoid cell death. The ROS-induced mechanism contributing to OXPHOS downregulation is probably one of many parallel mechanisms that take place in cancer cell proliferation during hypoxia adaptation.


   Supplementary material
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
Funding
 References
 
Supplementary Tables 1 and 2 can be found at http://carcin.oxfordjournals.org/


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
References
 
Ligue Régionale Contre le Cancer; Centre National de la Recherche Scientifique (UMR 5534, IFR 41); Institut National de la Santé et de Recherche Médicale (ATC VIE0208); Claude Bernard University of Lyon 1 (BQR grant); MSMT CR (MSM0021620806, 1M6837805002); GACR (303/07/0781).


   Footnotes
 
{dagger} These authors contributed equally to this work. Back


   Acknowledgments
 
We thank EGIDE for granting subsidies for travels between France and Czech Republic.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
 References
 

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Received December 29, 2007; revised April 26, 2008; accepted May 14, 2008.


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