Role of nitric oxide in the regulation of HIF-1alpha expression during hypoxia

Faton H. Agani, Michelle Puchowicz, Juan Carlos Chavez, Paola Pichiule, and Joseph LaManna

Department of Anatomy, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4938


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Hypoxia-inducible factor-1 (HIF-1), a heterodimeric transcription factor consisting of HIF-1alpha and HIF-1beta subunits, controls the expression of a large number of genes involved in the regulation of cellular responses to reduced oxygen availability. The oxygen-regulated subunit, HIF-1alpha , is stabilized in cells exposed to hypoxia. The regulation of hypoxic responses by nitric oxide (NO) is believed to have wide pathophysiological relevance, thus we investigated whether NO affects HIF-1 activation in hypoxic cells. Here we show that NO generated from NO donors prevented HIF-1alpha hypoxic accumulation in Hep 3B and PC-12 cells. Addition of a glutathione analog or peroxynitrite scavengers prevented the NO-induced inhibition of HIF-1alpha accumulation in both cell lines. Exposure to NO was associated with inhibition of mitochondrial electron transport and compensatory glycolysis, which maintained normal cellular ATP content. Succinate, a Krebs cycle intermediate and respiratory chain substrate, restored HIF-1alpha hypoxic induction in the cells, suggesting involvement of mitochondria in regulation of HIF-1alpha accumulation during hypoxia. Regulation of HIF-1alpha by NO is an additional important mechanism by which NO might modulate cellular responses to hypoxia in mammalian cells.

hypoxia-inducible factor-1; mitochondria; oxygen sensing


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

HYPOXIA-INDUCIBLE FACTOR-1 (HIF-1), a general hypoxia-inducible transcription factor, plays a central role in mediating cellular responses to hypoxia (reviewed in Refs. 41, 42, 51). HIF-1 was initially discovered through its ability to activate the erythropoietin gene during hypoxia via binding to the enhancer element located in the 3'-flanking region of the gene (43). Cloning of HIF-1 revealed a heterodimeric protein consisting of two subunits, HIF-1alpha and HIF-1beta (48, 50). HIF-1beta , identical to the aryl hydrocarbon receptor nuclear translocator, is a common binding subunit of many basic helix-loop-helix heterodimer transcription factors with Per-Arnt-Sim domains, besides HIF-1alpha (21). HIF-1beta is constitutively expressed, whereas HIF-1alpha is the oxygen-regulated subunit, rapidly accumulating in cells exposed to hypoxia (22). Under well-oxygenated conditions, the HIF-1alpha protein undergoes rapid ubiquitination and degradation by the proteasome system (23, 28, 38). HIF-1alpha is ubiquitinated by the von Hippel-Lindau protein (pVHL) (34), which binds directly to the oxygen-dependent degradation domain of HIF-1alpha and targets it for proteasome degradation (34, 46). VHL is associated with elongins B and C, cullin-2, and likely other factors that constitute part of a multiprotein complex (35, 46).

Interaction between pVHL and a specific domain of the HIF-1alpha subunit was reported to be regulated through hydroxylation of a proline residue by an enzyme prolyl hydroxylase (15, 25, 26). Prolyl hydroxylases have a requirement for dioxygen, iron, and 2-oxoglutarate (25, 26). In hypoxic conditions prolyl hydroxylation of the HIF-1alpha subunit is suppressed, leading to stabilization of the protein. Models based on a putative hemeprotein with oxygen-sensing properties (18), NADPH oxidase activity, and reactive oxygen species formation (16) have been proposed earlier to explain the ability of cells to sense changes in the oxygen concentration. We (1) and others (10, 11) have recently suggested that signaling from mitochondria might contribute to the activation of HIF-1alpha during hypoxia.

HIF-1alpha activation can be modulated by various factors including nitric oxide (NO; reviewed in Ref. 42). The regulation of hypoxic responses by NO is believed to have wider pathophysiological relevance. NO has been implicated in developmental and physiological responses to hypoxia in Drosophila melanogaster (52). Physiological concentrations of NO inhibit respiratory complex IV (cytochrome-c oxidase) in a manner that is competitive with oxygen, and this has potential to modulate cellular respiration (reviewed in Ref. 9). Importantly, high concentrations of NO, which cause damage to mitochondria, are encountered in various pathologies associated with hypoxia (such as brain ischemia-reperfusion injury) (7); thus NO may directly affect the outcome in these diseases (reviewed in Ref. 6).

Regulation of HIF-1alpha by NO is an additional important mechanism by which NO might modulate cellular responses to hypoxia. Recent studies addressing regulation of HIF-1 by NO reveal a complex picture. NO was found to inhibit HIF-1 DNA-binding activity and hypoxia-inducible gene expression in hypoxic cells (32, 44). Inhibition of HIF-1alpha accumulation in hypoxic cells by NO was reported (24), but this was not corroborated by others (32, 44). NO is also reported to induce HIF-1alpha , HIF-1 DNA-binding activity, and expression of HIF-1 target genes under nonhypoxic conditions via mechanisms that differ from NO suppression of HIF-1 activation in hypoxia (29, 30, 37, 40).

HIF-1alpha protein accumulation is the initial and crucial step in the activation of HIF-1 during hypoxia. Thus we address here specifically whether NO prevents HIF-1alpha protein accumulation in hypoxic cells and look for the possible involvement of mitochondria in mediating the effect of NO.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture and reagents. A human hepatoblastoma cell line Hep 3B (ATCC HB-8064) was maintained in MEM supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. PC-12 cells (ATCC CRL-1721) were grown in RPMI 1640 medium, supplemented with 10% fetal bovine serum, 5% horse serum, and 1% penicillin/streptomycin. All media and antibiotics were purchased from Life Technologies. The NO donor 2,2-(hydroxynitrosohydrazino)bis-ethanamine (DETA-NO; NOC-18) and glutathione ethyl ester (GSH-ethyl ester) were obtained from Calbiochem (San Diego, CA). S-nitroso-N-acetylpenicillamine (SNAP), and other reagents were purchased from Sigma (St. Louis, MO). Cells were pretreated with vehicle control or indicated agents for 1.5 h before exposure to hypoxia (1% O2-5% CO2-94% N2) for 4 h in Plexiglas modular chambers (Billups, Rothenburg). When indicated, cells were permeabilized with 4 µg/ml digitonin in a medium containing 4 mM succinate.

NO measurements. Concentrations of NO produced by DETA-NO and SNAP were measured using modified differential pulse voltametry as previously described (14). The electrochemical measurements were performed with BAS (BioAnalytical Systems) 100B/W and CH 660 (CH Instruments) electrochemical analyzers. A platinum wire (99.99%, Goodfellow) with a diameter of 25-75 µm was sealed into a glass capillary. To prepare standard NO stock solutions, NO was generated chemically from KNO2 by an excess amount of ascorbic acid, which reduces nitrite to NO (14). A solution containing (in mM) 30 KNO2, 30 HCl, and 90 NaCl was purged with nitrogen for 15 min in a rubber-sealed glass vial to remove traces of oxygen, followed by the addition of 30 mM crystalline ascorbic acid. Before adding NO donor, the cells were washed and medium was replaced with a balanced buffer (pH 7.0) containing (in mM) 108 NaCl, 5 KCl, 1.5 CaCl2, 0.5 MgCl2, 0.5 KH2PO4, 0.5 Na2HPO4, 25 HEPES, and 5 glucose. Cells were grown to confluency in 35-mm dishes, and the electrode for NO measurements was placed ~1 mm above the attached cells. Data analysis and numerical procedures were carried out with SigmaPlot 3.0 (Jandel Scientific).

Immunoblot analysis. To determine HIF-1alpha protein levels in cultured cells, nuclear extracts were prepared and 20 µg of nuclear protein per lane were used for electrophoresis. Membranes were blocked with 5% nonfat dry milk, incubated with monoclonal anti-HIF-1alpha antibodies either from Transduction Laboratories (Lexington, KY ) or Novus Biologicals (Littleton, CO), followed by secondary antibody detection by enhanced chemiluminescence (Amersham, Piscataway, NJ).

Nuclear extracts and DNA electrophoretic mobility shift assay. After treatment, cells were scraped into cold PBS, centrifuged, and washed in five packed cell volumes of buffer containing (in mM) 10 Tris · HCl (pH 7.5), 1.5 MgCl2, and 10 KCl and freshly supplemented with dithiothreitol (DTT), sodium vanadate, phenylmethylsulfonyl fluoride (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 (49). Nuclei were pelleted by centrifugation at 10,000 g for 10 min, the supernatant was discarded, and nuclei were resuspended in a buffer [0.42 M KCl, 20 mM Tris · HCl (pH 7.5), 20% (vol/vol) glycerol, and 1.5 mM MgCl2], freshly supplemented with DTT, sodium vanadate, PMSF, leupeptin, and aprotinin. The suspension was rotated at 4°C for 30 min and centrifuged for 30 min at 14,000 rpm. The supernatant containing the nuclear proteins was collected. Nuclear proteins were used for an electrophoretic mobility shift assay (49), using oligonucleotide probe from the erythropoietin enhancer region, which includes the HIF-1 binding site (5'-GCCCTACGTGCTGTCTCA-3').

Measurement of complex I activity. Mitochondria from cultured cells were prepared by discontinuous Percoll gradient centrifugation (2). Adherent cells were harvested and homogenized in an isolation buffer containing 0.32 M sucrose, 1 mM EDTA (K+ salt), and 10 mM Tris · HCl (pH 7.4). The homogenate was centrifuged at 1,330 g for 3 min and the supernatant recentrifuged at 21,200 g for 10 min. Collected mitochondria were isolated in a Percoll gradient. Complex I activity was measured as described previously (1), by measuring the oxidation of NADH at 340 nm using decylubiquinone as the electron acceptor. A total of 20-40 µg of mitochondrial protein were used, and assays were performed at 37°C in a Beckman DU 640 spectrophotometer. Data were reported as means ± SD. Statistical analysis between multiple groups was performed by one-way ANOVA with Tukey correction, and P < 0.05 was considered significant.

Cell viability analysis. After cells were exposed for 5.5 h to NO donors, cell viability was estimated by trypan blue exclusion (0.4% trypan blue, light microscopy) and lactate dehydrogenase (LDH) release in the medium (5). LDH release into the medium following exposure to 40 µg/ml of digitonin was taken as 100% of total activity.

ATP and lactate assays. Intracellular concentrations of ATP and lactate released in the media were assayed. Lactate was measured using a lactate analyzer (YSI 2300, STAT Plus; Yellow Springs Instrument, Yellow Springs, OH). For ATP measurements, Hep 3B and PC-12 cells were washed with PBS and lysed by adding 0.1M NaOH. The cells and media were collected, rapidly frozen, and stored at -80°C. ATP intracellular concentrations were analyzed using a luciferase bioluminescence procedure previously described (33). Briefly, 10 µl of cell extract were mixed with 50 µl of ATP reagent [solution mixture: 0.5 ml of imidazole-HCl buffer (1 M), 0.02 ml of MgCl2 (1 M), and 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 Chrono-log Lumi-Vette luminometer S900 (Chrono-log, Havertown, PA). All experiments were performed in triplicates, and the ATP content in nanomoles was normalized for cell protein. The protein content was measured using a standard Bradford assay (Bio-Rad).


    RESULTS
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SNAP prevents HIF-1alpha protein accumulation in Hep 3B and PC-12 cells during hypoxia. Here we show that incubation of Hep 3B and PC-12 cells with SNAP prevented hypoxic accumulation of HIF-1alpha (Fig. 1, A and B). At the concentration of SNAP used (50 µM), a concentration of 0.46 ± 0.1 µM NO (means ± SD, n = 3) was achieved at the end of the experiment (5.5 h of exposure). NO and its derivative peroxynitrite (ONOO-) can interact with a large number of proteins to modulate various cellular functions, including cell respiration (19). Incubation of cells with NO donors is reported to result in a rapid and reversible inhibition of cytochrome-c oxidase (complex IV), which can be reversed by removing NO (4, 8, 13). After prolonged incubation, cells show an additional inhibition of respiration, which appears to be due to inhibition of complex I (4, 8, 35, 36) and could be prevented or reversed by thiols (such as GSH-ethyl ester) or exposure to "cold" light (illumination from a halogen bulb) (4, 8).


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Fig. 1.   Effect of S-nitroso-N-acetyl-penicillamine (SNAP) on hypoxia-inducible factor-1alpha (HIF-1alpha ) protein levels in Hep 3B and PC-12 cells during hypoxia. Immunoblot of nuclear extracts prepared from Hep 3B and PC-12 cells pretreated for 1.5 h with SNAP, GSH-ethyl ester (GSH), or uric acid (UA), followed by 4-h exposure to hypoxia (1% O2). A: SNAP (50 µM) completely prevented HIF-1alpha expression in hypoxic Hep 3B cells. Including 2 mM GSH-ethyl ester or 1 mM uric acid in culture medium restored the hypoxic induction of HIF-1alpha . B: similarly, treatment of PC-12 cells with 50 µM SNAP completely prevented HIF-1alpha expression during hypoxia; 2 mM GSH-ethyl ester or 1 mM uric acid partially restored the hypoxic induction of HIF-1alpha . C: neither 2 mM GSH-ethyl ester nor 1 mM uric acid had any effect on HIF-1alpha expression levels under hypoxic or normoxic conditions in Hep 3B and PC-12 cells.

Excessive NO causes a decrease in cellular GSH levels (12, 27), which was reported to result in selective inhibition of complex I activity, and replenishing GSH content prevented the decrease in complex I activity (4, 12, 27). Thus, if NO-induced suppression of HIF-1alpha accumulation is mediated via primary inhibition of complex I activity, replenishing GSH might restore hypoxic induction of HIF-1alpha via its specific protective effects on complex I. When cells were incubated with SNAP together with 2 mM GSH-ethyl ester, hypoxic accumulation of HIF-1alpha was restored in Hep 3B cells (Fig. 1A) and partially restored in PC-12 cells (Fig. 1B). NO reacts rapidly with superoxide anion to form peroxynitrite, a strong cellular oxidant, which causes protein oxidation (reviewed in Ref. 19). We looked for the possible involvement of peroxynitrite species in suppression of HIF-1alpha hypoxic accumulation by incubating cells with uric acid, a scavenger of peroxynitrite. When uric acid was present in the medium, hypoxic stabilization of HIF-1alpha was restored in Hep 3B cells (Fig. 1A) and partially restored in PC-12 cells (Fig. 1B) treated with SNAP, suggesting that suppression of HIF-1alpha hypoxic accumulation may be due to peroxynitrite modification of target proteins. To exclude the possibility that GSH-ethyl ester or uric acid may alter HIF-1alpha protein levels in the absence of NO donor, we further tested the effects of these two agents under normoxic and hypoxic conditions in both cell lines (Fig. 1C). No effect on HIF-1alpha accumulation by either agent could be observed (Fig. 1C). Taken together, these results show that NO generated from NO donors inhibits HIF-1alpha accumulation in hypoxic cells. This effect of NO can be markedly prevented by maintaining cellular GSH content or by scavenging peroxynitrite.

DETA-NO (NOC-18) prevents HIF-1alpha accumulation in Hep 3B and PC-12 cells during hypoxia. DETA-NO, a chemically different agent from SNAP, is a slow-releasing NO donor (half-life of NO release approx  20 h) characterized by a controlled and spontaneous release of NO in solution. At the concentration of DETA-NO used (1 mM), a concentration of 1.48 ± 0.12 µM NO (means ± SD, n = 3) was achieved at the end of the experiment (5.5 h of exposure). DETA-NO effectively blocked accumulation of HIF-1alpha in hypoxic Hep 3B (Fig. 2A) and PC-12 cells (Fig. 2B). Thus the effects of SNAP can be reproduced by using a different NO donor, further confirming that release of NO is specifically responsible for suppression of HIF-1alpha during hypoxia. In agreement with the results obtained with SNAP, hypoxic Hep 3B and PC-12 cells (Fig. 2, A and B, respectively) treated with DETA-NO regained the ability to induce HIF-1alpha in the presence of GSH-ethyl ester (2 mM). These results confirm that excessive NO prevents HIF-1alpha accumulation in hypoxic cells.


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Fig. 2.   Effect of DETA-NO on HIF-1alpha protein levels in Hep 3B and PC-12 cells during hypoxia. A: immunoblot of nuclear extracts prepared from Hep 3B cells pretreated for 1.5 h with 2,2-(hydroxynitrosohydrazino)bis-ethanamine (DETA-NO; 1 mM) or GSH-ethyl ester (2 mM), followed by exposure to hypoxia for 4 h. DETA-NO completely inhibited HIF-1alpha induction in hypoxic Hep 3B cells; GSH-ethyl ester (G) restored HIF-1alpha induction. B: DETA-NO effectively prevented HIF-1alpha induction in hypoxic PC-12 cells; HIF-1alpha induction was partially restored by GSH-ethyl ester. C: Hep 3B cells were pretreated for 1.5 h with 1 mM DETA-NO or 20 mM methionine (METH), followed by 4 h exposure to hypoxia. Methionine restored HIF-1alpha hypoxic expression in DETA-NO-treated cells and had no effect on HIF-1alpha expression in normoxic or hypoxic cells that were not treated with DETA-NO. D: Hep 3B cells were pretreated for 1.5 h with 1 mM DETA-NO or 0.75 mM dithiothreitol (DTT), followed by exposure to hypoxia or normoxia for 4 h. DTT restored hypoxic expression of HIF-1alpha in DETA-NO-treated cells and had no significant effect on hypoxic or normoxic expression in cells that were not treated with DETA-NO.

Concerns regarding the lack of specificity may apply to the use of uric acid as a peroxynitrite scavenger. Thus we further tested methionine, another commonly used peroxynitrite scavenger. Figure 2C shows that methionine (20 mM) was also able to restore hypoxic induction of HIF-1alpha in DETA-NO-treated cells. Methionine did not affect HIF-1alpha normoxic or hypoxic levels (Fig. 2C).

GSH probably acts by replenishing the glutathione pool, but it may have other nonspecific effects; therefore, we next tested whether a thiol reducing agent would also be successful in restoring the hypoxic response. Indeed, 0.75 mM DTT was also able to restore HIF-1alpha hypoxic induction of DETA-NO-treated cells (Fig. 2D). DTT was also previously shown to prevent complex I inhibition by DETA-NO like GSH (8). DTT did not interfere with HIF-1alpha normoxic or hypoxic expression.

Effects of NO, GSH-ethyl ester, and uric acid on mitochondrial complex I activity. The mitochondrial model of oxygen sensing proposed by Schumacker and colleagues (10, 11) assumes that inhibition of electron flow at complex I prevents hypoxic accumulation of HIF-1alpha . Thus any significant inhibition of complex I by NO would be expected to prevent HIF-1alpha hypoxic accumulation. This prompted us to examine complex I activity in NO-treated cells.

To determine the extent of complex I inhibition by NO, the enzyme activity was assayed in mitochondrial homogenates from Hep 3B and PC-12 cells treated with SNAP and DETA-NO (Fig. 3, A and B).


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Fig. 3.   Effect of SNAP, DETA-NO, GSH-ethyl ester, and uric acid on mitochondrial complex I activity in Hep 3B and PC-12 cells. Complex I activity (nmol · min-1 · mg protein-1) was assayed in mitochondrial homogenates from cells treated with SNAP (50 µM, 5.5 h) or DETA-NO (1 mM, 5.5 h), in the presence or absence of GSH-ethyl ester (2 mM) or uric acid (1 mM). Statistical analysis was performed by one-way ANOVA (means ± SD; asignificantly lower than complex I activity in control cells; bsignificantly higher than complex I activity in DETA-NO-treated cells; csignificantly higher than complex I activity in SNAP-treated cells). A: changes in complex I activity in Hep 3B cells. B: changes in complex I activity in PC-12 cells.

Complex I activity in untreated Hep 3B cells (141 ± 29; n = 6) decreased by 74% after DETA-NO (36 ± 5; n = 3), compared with 86% inhibition by 1 µM rotenone (20 ± 4, n = 3) (Fig. 3A). This DETA-NO inhibition of complex I was almost completely prevented in the presence of GSH-ethyl ester (15% inhibition; 120 ± 12; n = 3; Fig. 3A).Complex I activity in untreated PC-12 cells (138 ± 11; n = 9) decreased by 52% after SNAP (66 ± 5; n = 3), compared with 88% inhibition by 1 µM rotenone (16 ± 3; n = 3; Fig. 3B). SNAP-induced inhibition was significantly attenuated by GSH-ethyl ester, resulting in only 17% complex I inhibition (115 ± 4; n = 3), and significantly attenuated by uric acid, resulting in 31% complex I inhibition (95 ± 15; n = 3; Fig. 3B). Similarly, DETA-NO strongly inhibited enzyme activity (70% complex I inhibition; 41 ± 10; n = 3), which was almost completely counteracted in the presence of GSH-ethyl ester (9% complex I inhibition; 125 ± 8; n = 3; Fig. 3B).

Thus 5.5 h of exposure to SNAP caused a 52% decrease in complex I activity in PC-12, while DETA-NO caused a 74% complex I inhibition in Hep 3B cells and 70% inhibition in PC-12 cells, which is comparable to the effect of rotenone, a specific and potent complex I inhibitor, previously shown to inhibit HIF-1alpha hypoxic induction (1, 10, 11). Taken together, these results show that NO-induced inhibition of complex I is severe and could provide a possible explanation for NO suppression of HIF-1alpha accumulation according to mitochondrial model of HIF-1 regulation (10, 11).

Because GSH-ethyl ester and uric acid restored HIF-1alpha induction in SNAP and DETA-NO-treated cells during hypoxia (Figs. 1 and 2), we tested whether they can prevent the decrease in complex I activity. GSH-ethyl ester prevented nearly completely the inhibition of complex I, while uric acid attenuated significantly the decrease in complex I activity by SNAP in PC-12 cells (Fig. 3B). Similarly, incubation with GSH-ethyl ester prevented the decrease in complex I activity by DETA-NO in Hep 3B cells (Fig. 3A). These results show that NO-induced complex I inhibition is significantly attenuated by GSH-ethyl ester and uric acid, which correlates with their ability to restore HIF-1alpha hypoxic induction in these cells.

Succinate, a Krebs cycle intermediate and complex II respiratory substrate, overcomes NO suppression of HIF-1alpha stabilization in hypoxic cells. Inhibition of mitochondrial complex I (1, 10, 11), as well as complex III (at the myxothiazol binding site) (10, 11), but not inhibition of complex IV (41), was reported to prevent induction of HIF-1alpha in hypoxic cells. Thus electron flux into complex III was suggested to be necessary for normal HIF-1alpha accumulation during hypoxia (10, 11). If NO prevents HIF-1alpha hypoxic accumulation by inhibiting complex I activity, then NO suppression should be overcome by bypassing complex I and using complex II as an alternative route of electron transport into complex III. In this experiment succinate was used as a complex II substrate in digitonin-permeabilized cells. In both Hep 3B (Fig. 4A) and PC-12 (Fig. 4B) cells, DETA-NO prevented accumulation of HIF-1alpha during hypoxia. However, DETA-NO suppression of HIF-1alpha was overcome in the presence of succinate (4 mM) and digitonin (4 µg/ml) in both cell lines (Fig. 4, A and B). We also assayed HIF-1 DNA binding in nuclear extracts from PC-12 cells. As expected, HIF-1 DNA binding paralleled the increase in the HIF-1alpha protein levels after hypoxia, DETA-NO, and succinate treatments (Fig. 4B). To exclude the possibility that digitonin or succinate may alter HIF-1alpha protein levels, we tested the possible effects of these two agents in the absence of NO donors (Fig. 4C). Digitonin, either alone or combined with succinate, had no effect on normoxic or hypoxic protein levels of HIF-1alpha (Fig. 4C). Thus NO-treated hypoxic Hep 3B and PC-12 cells regained the ability to induce HIF-1alpha in the presence of succinate, which maintains electron flow via complex II into complex III. These results show that complex I is targeted by NO and suggest that maintaining electron transport chain activity into complex III is necessary for hypoxic induction of HIF-1alpha .


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Fig. 4.   Effect of succinate on HIF-1alpha induction and HIF-1 DNA binding in DETA-NO-treated Hep 3B and PC-12 cells during hypoxia. Nuclear extracts were prepared from Hep 3B and PC-12 cells pretreated with DETA-NO (1 mM) for 1.5 h, in the presence or absence of succinate (S; 4 mM) and digitonin (D; 4 µg/ml), followed by 4-h exposure to hypoxia (1% O2). A: DETA-NO suppression of HIF-1alpha induction in hypoxic Hep 3B cells was restored by succinate and digitonin. B: presence of succinate (4 mM) and digitonin (4 µg/ml) in the medium restored both HIF-1alpha protein induction and HIF-1 DNA binding in DETA-NO-treated hypoxic PC-12 cells. C, constitutive binding. C: digitonin (4 µg/ml), or combined digitonin plus succinate (4 mM) treatment, had no effect on HIF-1alpha expression levels under normoxic or hypoxic conditions in Hep 3B and PC-12 cells after 5.5 h of treatment.

To minimize the toxicity of digitonin to cultured cells, the standard digitonin protocol was modified (31), and a low dose (4 µg/ml) of digitonin was used in all experiments. This concentration of digitonin did not alter the viability of Hep 3B or PC-12 cells throughout 5.5 h of exposure in our experiments, as determined by trypan blue exclusion (Fig. 5A) or LDH release in the medium (Fig. 5B). Moreover, we observed no effect on cell viability by trypan blue in the presence of both digitonin and either NO donor for 5.5 h of exposure (Fig. 5A). To gain a better insight into metabolic effects of the treatment, we further examined the changes in the ATP and lactate levels in both cell lines (Fig. 6). Exposure to digitonin for 5.5 h did not affect cellular ATP and lactate content, consistent with the notion that a low dose of digitonin in our experiments does not significantly affect cell homeostasis (Fig. 6). Exposure to NO donors did not significantly alter cellular ATP content in the cells but resulted in increased lactate production, an anticipated outcome of respiratory chain inhibition by NO, which leads to compensatory glycolysis (3) (Fig. 6). Similarly, hypoxia caused a comparable increase in lactate production (Fig. 6), while combined NO and hypoxia did not cause any further elevation in lactate compared with each treatment alone (data not shown). These results suggest that both cell lines have high glycolytic capacity and were able to maintain ATP levels throughout 5.5 h of duration of the experiment through increased glycolysis, which is in agreement with a previous report (3).


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Fig. 5.   Viability of Hep 3B and PC-12 cells is not altered by treatment procedures. A: cell viability testing by trypan blue exclusion in Hep 3B and PC-12 cells treated for 5.5 h with 4 µg/ml digitonin, 1 mM DETA-NO, or 50 µM SNAP, or untreated (n = 3 for each treatment group; means ± SD). B: lactate dehydrogenase (LDH) release in the medium was determined in cells treated for 5.5 h with 4 µg/ml digitonin or a high dose of 40 µg/ml (10×) digitonin (n = 3 in each treatment group). LDH release into the medium following exposure to the high dose of digitonin (40 µg/ml), which resulted in cell death, was taken as 100% of total activity.



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Fig. 6.   Changes in ATP and lactate levels after exposure to NO donors. Intracellular ATP levels (nmol/mg protein) and lactate released in the medium (mM) were determined in Hep 3B and PC-12 cells after 5.5 h of treatment with 4 µg/ml digitonin, 1 mM DETA-NO, 50 µM SNAP, hypoxia, and in untreated cells (n = 3 in each treatment group; means ± SD; * P < 0.05, Student's t-test).

Effects of DETA-NO and SNAP on normoxic induction of HIF-1alpha protein levels in normoxic Hep 3B and PC-12 cells. In view of recent reports that NO induces HIF-1alpha during normoxia (29, 30, 37, 39), we tested the effects of DETA-NO and SNAP in normoxic Hep 3B and PC-12 cells (Fig. 7, A and B, respectively). DETA-NO (1 mM) induced only minimal and transient HIF-1alpha protein expression in normoxic Hep 3B cells after 5.5 and 8 h (Fig. 7A, lanes 3 and 4), but not in PC-12 cells (Fig. 7B, lanes 3 and 4). No induction of HIF-1alpha during normoxia could be demonstrated with SNAP (50 µM) in either cell line (Fig. 7, A and B, lanes 5 and 6). Therefore, under conditions tested, we observed only a minor induction of HIF-1alpha by DETA-NO in normoxic Hep 3B cells.


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Fig. 7.   Effect of SNAP and DETA-NO on normoxic HIF-1alpha induction in Hep 3B and PC-12 cells. Immunoblots are shown of nuclear extracts from Hep 3B (A) and PC-12 cells (B) treated with NO donors. Cells were left untreated (lane 1), exposed to hypoxia (1% O2, 4 h; lane 2), treated with 1 mM DETA-NO for 5.5 or 8 h under normoxic conditions (lanes 3 and 4, respectively), or treated with 50 µM SNAP for 5.5 or 8 h under normoxic conditions (lanes 5 and 6, respectively). A minimal normoxic induction of HIF-1alpha protein was observed only in Hep 3B cells treated with DETA-NO (A, lanes 3 and 4).


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Current attempts to understand the molecular basis of cellular oxygen sensing are focused on the regulation of HIF-1alpha and the key question: How is the decrease in oxygen concentration sensed by the cells and what signaling pathways lead to HIF-1alpha protein stabilization? An important development are the recent reports of HIF-1alpha regulation via direct proline hydroxylation by an enzyme prolyl hydroxylase, which requires molecular oxygen, iron, and 2-oxoglutarate for enzymatic activity (15, 25, 26). The requirement for molecular oxygen and iron for proline hydroxylase activity could explain the stabilization of HIF-1alpha under hypoxic conditions or after treatment with agents that eliminate or compete with iron. However, the process of oxygen sensing is likely more complex. Earlier studies suggested that oxygen sensing involves reactive oxygen species, protein phosphorylation, signaling from mitochondria, and activation of multiple signal transduction pathways (reviewed in Refs. 41, 42, 51). Conceivably, these changes could directly or indirectly affect various steps of HIF-1alpha activation, including proline hydroxylation, although the connections are not clear at present.

Although several recent reports agree that NO inhibits HIF-1 DNA binding and transcriptional activation of HIF-1 target genes (24, 32, 44), there appears to be no consensus when it comes to the ability of NO to prevent HIF-1alpha accumulation during hypoxia. Our results show that excessive NO abrogates HIF-1alpha accumulation in hypoxic cells. To explain the mechanisms of NO suppression of HIF-1alpha activation, it was suggested that NO might interact with a putative oxygen sensor (24). Importantly, HIF-1alpha itself could be the target of S-nitrosylation; however, an earlier study found no evidence for direct modification of HIF-1alpha by NO, making this an unlikely explanation (24).

Herein we tested an alternative explanation for the NO suppression of HIF-1 that involves the primary inhibition of the mitochondrial electron transport by NO. Physiological and reversible inhibition of complex IV by NO is an early event. However, with time, a pathological inhibition/damage of complex I is reported to occur (4, 8). Our study shows that both SNAP and DETA-NO caused a marked 52 and 70-74% complex I inhibition, respectively. Thus, on the basis of the mitochondrial model of HIF-1 regulation (10, 11), the potent inhibitory effect of NO on complex I could explain its ability to block hypoxic accumulation of HIF-1alpha . This appears to be further indirectly supported by the findings that 1) addition of GSH-ethyl ester prevented the decrease of complex I activity by NO and restored HIF-1alpha hypoxic accumulation and 2) uric acid, a scavenger of peroxynitrite, markedly attenuated the inhibition of complex I activity and restored HIF-1alpha hypoxic induction in the presence of NO. These findings show a correlation between complex I inhibition and impaired HIF-1alpha hypoxic accumulation.

NO suppression of HIF-1alpha hypoxic accumulation could be overcome by the complex II substrate succinate, which restores electron flow into complex III, indicating an isolated inhibition/damage of complex I by NO. Under these experimental conditions, NO should also inhibit complex IV (reviewed in Ref. 9), thus succinate does not fully restore electron transport. However, complex IV inhibition was reported to have no effect on HIF-1alpha hypoxic induction (41). Again, these findings seem to be in agreement with the proposed model according to which the mitochondrial signal for HIF-1alpha stabilization is generated at complex III (10, 11), although a possible alternative explanation emerges from recently reported studies (25, 26, 45, 47). The requirement for an active mitochondrial electron transport chain in hypoxic HIF-1alpha accumulation was very recently questioned in two reports, which show that HIF-1alpha protein stabilization and transcriptional activity were preserved in cells lacking mitochondrial DNA (rho 0 cells) (45, 47). In these two studies, the HIF-1alpha response was tested in cells exposed to 0.1% O2 (nearly anoxia) (47) and 0.5% O2 (45), rather than 1.5% O2 (10, 11) or 1% (1, 20). If prolyl hydroxylases require oxygen as a substrate, then severe oxygen deprivation close to anoxia should cause HIF-1alpha stabilization, because in the virtual absence of oxygen the proline hydroxylation would always be inhibited. Hence, the fact that anoxia (or near anoxia) stabilizes HIF-1alpha in cells does not exclude the possibility that mitochondrial complex III might be required for a HIF-1alpha response toward a less severe oxygen deprivation (hypoxia). In support of the mitochondrial model, it was previously reported that pharmacological inhibitors of complex I rotenone (1, 10, 11, 20), 1-methyl-4-phenylpiridinium (1), diphenylene iodonium (10, 11), and complex III inhibitor myxothiazol (10, 11) prevent hypoxic stabilization of HIF-1alpha . Clearly, further studies are required to resolve the existing controversies and to delineate the role of the respiratory chain in HIF-1 regulation. These results, however, do not rule out the possible involvement of the mitochondria in regulation of HIF-1 via different mechanisms. For example, a novel prolyl hydroxylase was shown to regulate HIF-1alpha degradation/stabilization (25, 26). Prolyl hydroxylases require the Krebs cycle intermediate 2-oxoglutarate (alpha -ketoglutarate) as cosubstrate (25, 26). It was shown that synthesized structural analogs of 2-oxoglutarate can interfere with HIF-1alpha protein induction (26). Thus perturbations in Krebs cycle metabolism may possibly have a potential to interfere with regulation of HIF-1alpha . The ability of succinate to restore HIF-1alpha induction thus may hint toward an alternative explanation that metabolic effects of Krebs cycle intermediates, rather than restoration of electron transport, might impinge on signaling pathways that lead to HIF-1alpha accumulation under hypoxic conditions. This appears intriguing, because it could reconcile the role of mitochondria with the model of oxygen-dependent prolyl hydroxylation of HIF-1alpha .

In view of the recent intriguing reports that NO induces HIF-1alpha under normoxic conditions, we tested the effect of SNAP and DETA-NO in normoxic Hep 3B and PC-12 cells. We observed a minimal induction of HIF-1alpha only in Hep 3B cells after DETA-NO, and no effect of SNAP in either cell line (Fig. 7, A and B). Although the use of different NO donors, dose, length of treatment, and cell type-specific responses may account for some of the observed differences between this and previous studies, the issue may be more complex (29, 30, 37, 39). Reactive oxygen and nitrogen species might each modulate HIF-1alpha expression in hypoxic cells via distinct mechanisms (17). Recently, it was suggested that distinct cis elements in the promotor of some genes (such as the vascular endothelial factor) bind protein complexes and mediate gene induction by NO under normoxic conditions (30). The mechanisms by which NO suppresses HIF-1alpha hypoxic stabilization thus differ from the activation of HIF-1alpha in normoxic cells.

In conclusion, we show that NO interferes with the signaling events that lead to HIF-1alpha accumulation during hypoxia. This may have important consequences, because a number of pathologies associated with oxygen deprivation are also linked to excessive NO production. NO suppression of HIF-1alpha hypoxic accumulation was counteracted by the Krebs cycle intermediate succinate. This suggests that succinate acts by restoring electron flow into respiratory complex III. Alternatively, this may indicate an unsuspected metabolic effect of succinate, which hints toward a link between the Krebs cycle and regulation of HIF-1alpha under hypoxic conditions.


    ACKNOWLEDGEMENTS

We thank Miklos Gratzel and Gautham Shetty for help with nitric oxide measurements and Sue Foss and Max Neal for preparing and editing the manuscript.


    FOOTNOTES

This work was supported by National Institute of Neurological Disorders and Stroke Grants NS-41309 (to F. H. Agani), and NS-37111 and NS-38632 (to J. LaManna). F. H. Agani is also supported by National Heart, Lung, and Blood Institute Grant HL-56470.

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.

First published February 20, 2002;10.1152/ajpcell.00381.2001

Received 7 August 2001; accepted in final form 18 February 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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