Inhibition of Hypoxia-inducible Factor 1 Activation by Carbon Monoxide and Nitric Oxide
IMPLICATIONS FOR OXYGEN SENSING AND SIGNALING*

L. Eric HuangDagger , William G. WillmoreDagger , Jie Gu, Mark A. Goldberg, and H. Franklin Bunn§

From the Division of Hematology, Brigham & Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

It has been proposed that cells sense hypoxia by a heme protein, which transmits a signal that activates the heterodimeric transcription factor hypoxia-inducible factor 1 (HIF-1), thereby inducing a number of physiologically relevant genes such as erythropoietin (Epo). We have investigated the mechanism by which two heme-binding ligands, carbon monoxide and nitric oxide, affect oxygen sensing and signaling. Two concentrations of CO (10 and 80%) suppressed the activation of HIF-1 and induction of Epo mRNA by hypoxia in a dose-dependent manner. In contrast, CO had no effect on the induction of HIF-1 activity and Epo expression by either cobalt chloride or the iron chelator desferrioxamine. The affinity of CO for the putative sensor was much lower than that of oxygen (Haldane coefficient, ~0.5). Parallel experiments were done with 100 µM sodium nitroprusside, a nitric oxide donor. Both NO and CO inhibited HIF-1 DNA binding by abrogating hypoxia-induced accumulation of HIF-1alpha protein. Moreover, both NO and CO specifically targeted the internal oxygen-dependent degradation domain of HIF-1alpha , and also repressed the C-terminal transactivation domain of HIF-1alpha . Thus, NO and CO act proximally, presumably as heme ligands binding to the oxygen sensor, whereas desferrioxamine and perhaps cobalt appear to act at a site downstream.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Adaptation to hypoxia is of fundamental importance in developmental, physiological, and pathophysiological processes (1, 2). Humans and other mammals respond to low oxygen tension in part by fine tuning the expression of a group of physiologically relevant genes. Erythropoietin (3, 4) and tyrosine hydroxylase (5) act in concert to raise blood oxygen levels by enhancing erythropoiesis and ventilation, respectively. Hypoxic induction of genes encoding vascular endothelial growth factor (6) and inducible nitric oxide synthase (7, 8) leads to increased angiogenesis and vasodilation. Up-regulation of genes encoding glucose transporters and specific glycolytic isoenzymes (9-11) maximize ATP production in the setting of reduced oxygen supply. Remarkably, the hypoxic induction of all of these diverse genes appears to depend on a common mode of oxygen sensing and signal transduction, triggering the activation of a critical transcription factor, hypoxia-inducible factor 1 (HIF-1)1 (for review, see Ref. 1).

HIF-1 is a widely expressed heterodimeric protein composed of HIF-1alpha and ARNT subunits, both of which belong to the basic helix-loop-helix periodic-arylhydrocarbon receptor-simultaneous family (12, 13). Its biological importance is underscored by recent observations that both HIF-1alpha (14, 15) and ARNT (16) null mutant mouse embryos exhibit developmental arrest and lethality resulting essentially from impairment of vascularization. At the mRNA level, both HIF-1alpha and ARNT genes are constitutively expressed and not significantly up-regulated by hypoxia (17-20). Whereas changes in oxygen tension fail to affect ARNT protein abundance (19, 20), hypoxia markedly increases levels of HIF-1alpha protein (13, 19, 20). In fact, in normoxic cells HIF-1 is barely detectable (21). Moreover HIF-1alpha is remarkably unstable, with a half-life of <5 min (19). Thus, hypoxia-induced activation of HIF-1 depends in part on posttranslational stabilization of HIF-1alpha . In addition, hypoxia markedly enhances transactivating function of HIF-1alpha (22-24). The oxygen-dependent degradation (ODD) of HIF-1alpha is governed by an internal 200-residue ODD domain (25) via the ubiquitin-proteasome pathway (25, 26). Removal of the ODD domain renders HIF-1alpha stable under normoxia, resulting in autonomous heterodimerization, DNA binding, and transactivation in the absence of hypoxia signaling (25).

The activation of HIF-1 by hypoxia depends on a sensing and signaling process that is poorly understood. There is strong, albeit circumstantial, evidence that the oxygen sensor is a heme protein (1, 27). Support for this claim derives largely from experiments demonstrating that heme binding ligands suppress the hypoxic induction of HIF-1 activation and expression of the genes mentioned above. The hypoxic induction of erythropoietin (27), vascular endothelial growth factor (28, 29), and other genes (30) was markedly blunted in the presence of carbon monoxide (CO), a molecule whose only established biological function is binding to ferrous atoms on heme groups. Subquently, another heme ligand, nitric oxide (NO), was shown to exert a similar effect (29, 31, 32). In addition to hypoxia, HIF-1 can be activated by the transition metal Co2+ as well as by iron chelation (33). The mechanisms underlying the activities of these agonists are unknown, although there is circumstantial evidence that they affect the level of reactive oxygen species (34), which may serve as signaling molecules (1). To gain insight into hypoxic sensing and signaling, we have made an in-depth comparison of the downstream effects of CO and NO on HIF-1 activation, HIF-1alpha stability, and the expression of oxygen-responsive endogenous and reporter genes.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Reagents and Cell Culture-- Sodium nitroprusside (SNP), 8-bromo-cGMP, cobaltous chloride, and desferrioxamine were all purchased from Sigma.

Hep3B cells were incubated in alpha-modified Eagle's medium (Life Technologies, Inc.), and 293 cells were incubated in Dulbecco's modified Eagle's medium (Life Technologies, Inc.), supplemented with 10% fetal bovine serum (Hyclone), both at 37 °C. For experiments involving CO treatment, cells were seeded in 75-cm2 tissue culture flasks (Becton Dickinson, Franklin Lakes, NJ) fitted with autoclaved rubber stoppers punctured with sterile inflow (3.5-inch, 17-gauge) and outflow (1.5-inch, 18-gauge) needles. Gas flow was maintained at 300 ml/min. In some experiments, cobaltous chloride, desferrioxamine, or SNP was added to the culture medium at final concentrations of 100, 130, and 100 µM, respectively. Cells were exposed to one of the following gas mixtures: normoxia (95% air and 5% CO2), hypoxia (1% oxygen, 94% N2, and 5% CO2), normoxia with CO (21% O2, 10% CO, 5% CO2, and balance N2), or hypoxia with CO (1% O2, 10 or 80% CO, 5% CO2, and balance N2). Gases were mixed with a Series 200 computerized gas standards generator (Environics, Tolland, CT) and humidified by being passed through 0.15 M NaCl.

RNA Preparation and Analysis-- Total RNA was extracted by the TRIzol method (Life Technologies, Inc.) according to the manufacturer's protocol and dissolved in 100% formamide. A plasmid, containing a portion of exon 5 of the erythropoietin (Epo) gene (bp 2650-2890) inserted into pBluescript, was linearized with XhoI and in vitro transcribed in the presence of [32P]CTP (DuPont NEN) with T7 polymerase (Promega). RNase protection assays were performed with 40-100 µg of RNA hybridized at 55 °C overnight with the Epo riboprobe. For quantification of gels, a PhosphorImager was used with ImageQuant software (Molecular Dynamics, Sunnyvale, CA). Results are presented as the mean ± S.E. from independent experiments.

Plasmids-- Both p(HA)HIF-1alpha and p(HA)HIF-1alpha (401Delta 603) are (CMV)-driven expression vectors expressing hemagglutinin (HA)-tagged HIF-1alpha and the ODD domain-deleted HIF-1alpha (25). p(HA)HIF-1alpha (C520S) contains a replacement of cysteine at codon 520 with serine generated by the Altered Sites II in vitro mutagenesis system (Promega). pGal4-ODD and the human erythropoietin 3' enhancer reporter pEpoE-luc were constructed as described previously (19, 25). pGal4-luc and pSB92 (Gal4-CAD) were kindly provided by S. Bhattacharya from David Livingston's Laboratory.

Transient Transfection-- Human embryonic kidney 293 cells and human hepatoma Hep3B cells were transfected in suspension using the calcium phosphate method as described elsewhere (19). In general, 293 cells were transfected with 2 µg of each expression vector plus 0.25 µg of pCMV-beta gal, whereas Hep3B cells were transfected with 2 µg of pCMV-beta gal and 2 µg of reporter plasmid. Cells were treated with 100 µM sodium nitroprusside as indicated. After 24 h of incubation, whole cell extracts were prepared as described previously (19) for electrophoretic mobility shift assays and Western blotting. For reporter gene assays, cells were harvested 48 h after transfection and assayed for luciferase activity as described elsewhere (19).

Electrophoretic Mobility Shift Assay (EMSA) and Western Blotting-- Whole cell extracts were analyzed for HIF-1 or Gal4 DNA binding activity or were subjected to Western blot analysis as described previously (19, 25). Endogenous HIF-1alpha protein was detected by anti-HIF-1alpha monoclonal antibodies: a mix of monoclonal antibodies OZ12 and OZ15 directed against the C-terminal half (residues 530-826; Ref. 35) (kindly provided by Dr. David Livingston), monoclonal antibody 28b raised against residues 329-530 (36) (kindly provided by Dr. Patrick Maxwell), and anti-HIF-1alpha (Novus Biologicals, Inc., Littleton, CO. All three monoclonal antibodies gave internally consistent results. The monoclonal anti-HA 12CA5 (Boehringer Mannheim) antibody was used to determine HA-tagged HIF-1alpha expression. Polyclonal anti-ARNT antibodies were used as before (19). The antigen-antibody complexes were visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech).

Calculation of the Haldane Coefficient for Oxygen versus CO Binding-- To calculate the fractional saturation of the oxygen sensor with ligand (O2 versus CO), we used a comprehensive and accurate set of earlier experimental data from our laboratory (37) on the relationship between pO2 and level of Epo mRNA. These data, in combination with experimental quantitation of Epo mRNA, enabled the estimation of pOs values, the pO2 simulated by O2 + CO ligation. Because the relevant experiments were carried out in 1% O2 (=7 torr), the Haldane coefficient M was calculated from the equation: M = (pOs - 7)/COtorr.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Effect of Carbon Monoxide and Nitric Oxide on the Induction of Epo mRNA-- Of all the genes up-regulated by hypoxia, Epo shows the most robust response. Therefore we began by examining the effect of CO and NO on the induction of Epo mRNA in Hep3B cells. Epo mRNA, protected by a 240-bp riboprobe during ribonuclease protection assays, displayed a doublet banding pattern (Fig. 1, A and B). As expected, hypoxia, cobalt, and desferrioxamine markedly induced Epo mRNA expression. CO suppressed hypoxic induction of Epo mRNA, almost to the level of normoxic controls (Fig. 1A, lanes 3 and 4). In determining the Haldane coefficient for the oxygen sensor, we assumed that CO and O2 bind to the same site, similar to other heme proteins. At 10 and 80% CO, the calculated Haldane coefficients were 0.8 and 0.2, respectively. Interpretation of these values may be confounded by the possibility that, like cytochrome c oxidase, CO is likely to bind reversibly to the heme group of the oxygen sensor, whereas oxygen binding may be irreversible if it undergoes reduction to superoxide (1, 38). However, the coefficients that we obtained were reproducible and probably valid, owing to the high flow of gas used in the incubations and the likelihood that the pO2 was maintained at a constant level. Cobalt and hypoxia had an additive effect on Epo mRNA levels, whereas desferrioxamine and hypoxia did not. Combined cobalt and hypoxia induction was decreased by 10 and 80% CO treatment, to the levels of normoxic cobalt induction. Cobalt induction of Epo mRNA under normoxic conditions was not suppressed by 10% CO (Fig. 1A, lane 14). Likewise, CO had no effect on desferrioxamine-induced Epo mRNA levels (Fig. 1A, lane 11). These results indicate that CO does not suppress Epo mRNA expression in a nonspecific toxic manner.


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Fig. 1.   Effect of CO and NO on induction of Epo mRNA expression in Hep 3B cells. A, cells were exposed to 20% O2 (N) or 1% O2 (H) and incubated in either control medium (lanes 1-4) or in media containing 100 µM cobalt (lanes 5-8) or 130 µM desferrioxamine (lanes 9-11). Aliquots of cells were exposed to 10% carbon monoxide (lanes 3, 7, and 11-13), and 80% CO (lanes 4 and 8). Ribonuclease protection assays were performed using radiolabeled wild-type Epo riboprobe (250 bp of the fifth exon, which includes the 3' untranslated region). The bar graph below shows densitometric quantitation of replicate experiments: means ± S.E. of with the number of experiments indicated above each bar. B, experiments similar to A tested the effect of 100 µM SNP on induction of Epo mRNA by hypoxia and cobalt.

Parallel experiments were done with 100 µM SNP as a source of NO. As shown in Fig. 1B, lane 3, this concentration of SNP mimicked CO in suppressing the induction of Epo mRNA by hypoxia. However, in contrast to CO, SNP suppressed induction of Epo mRNA by cobalt (Fig. 2, lanes 5, 7, and 9). These results with SNP are consistent with those of Sogawa et al. (32), who tested a hypoxia-inducible reporter gene with SNP and two other NO donors.


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Fig. 2.   Effect of CO and NO on induction of HIF-1 binding and HIF-1alpha protein in Hep 3B cells. A and B, effect of CO; C and D, effect of SNP as a source of NO. A and C, results obtained on endogenous HIF-1 in Hep3B cells; B and D, results with overexpression of HIF-1alpha and ARNT subunits after transfection of p(HA)HIF-1alpha and pARNT into 293 cells. Cells were exposed to normoxia (N, 20% O2) or hypoxia (H, 1% O2) in the absence or presence of 10% CO, 80% CO, or 100 µM SNP. Cells were incubated either in control medium or in media containing 100 µM cobalt (lanes 2 and 4). Whole cell extracts were prepared 24 h after transfection and analyzed with EMSAs using 32P-labeled oligonucleotides containing an HIF-1 binding site. HIF-1, induced binding; C, constitutive binding; N, nonspecific binding; F, free probe. Shown below are Western blots of the same extracts used for the EMSAs, probed with monoclonal anti-HIF-1alpha antibody (A and C) or with anti-HA antibody (B and D).

Effect of CO and NO on the Activation of HIF-1-- To delineate the mechanism by which CO and NO suppressed Epo mRNA expression, we examined how these agents affected the activation of HIF-1, the transcription factor responsible for the regulation of Epo and a number of other genes induced by hypoxia. Extracts prepared from hypoxic cells, when incubated with radiolabeled wild-type HIF-1 binding sequence, displayed doublet bands in gel shift assays. In contrast, single bands were observed in extracts prepared from cells treated with cobalt (Fig. 2, lane 2) or desferrioxamine. Similar results have been reported by others (12, 33, 39). Hypoxia-induced HIF-1 binding was attenuated by treatment with CO in a dose-dependent manner (Fig. 2, lanes 5 and 6). In contrast, cobalt and desferrioxamine induction of HIF-1 binding was not affected by CO treatment (Fig. 2, lanes 7 and 8).

Similar to what was observed with CO, 100 µM SNP significantly suppressed hypoxia-induced HIF-1 DNA binding in Hep3B cells, whereas neither constitutive nor nonspecific binding was affected (Fig. 2, lane 14). To investigate the mechanism underlying the inhibitory effects of CO and SNP, 293 cells were transfected with the CMV-driven vectors overexpressing HIF-1 (HA-tagged HIF-1alpha and ARNT, respectively). Both CO and NO abrogated DNA binding of overexpressed HIF-1 (Fig. 2, lanes 11 and 17). It is noteworthy that under normoxia, despite exogenous overexpression of HIF-1, no HIF-1 binding activity was detected (Fig. 2, lanes 9 and 15). Together these results indicate that both endogenous and exogenous HIF-1 are regulated by the same signaling pathway.

Because HIF-1 activity is primarily determined by the abundance of HIF-1alpha protein (19), we examined by Western blot whether CO and NO affect HIF-1alpha accumulation in hypoxic cells. As shown in Fig. 2, bottom, exposure of hypoxic cells to 10 and 80% CO resulted in a dose-dependent reduction in the abundance of HIF-1alpha protein (Fig. 2, lanes 5 and 6), comparable with the decrease in HIF-1 DNA binding on EMSA. Importantly, CO had no effect on the abundance of HIF-1alpha induced by cobalt (Fig. 2, lanes 7 and 8), again indicating that CO was not exerting a nonspecific toxic effect. In a like manner, we examined whether NO affects HIF-1alpha accumulation in hypoxic cells. Consistent with the results obtained with CO, SNP blocked hypoxia-induced accumulation of endogenous HIF-1alpha (Fig. 2, lane 14). Moreover, both CO and NO suppressed accumulation of transfected HIF-1alpha (Fig. 2, lanes 11 and 17). In all these results, the level of HIF-1alpha closely paralleled that of HIF-1 binding activity.

The Oxygen-dependent Degradation Domain Is Responsible for Inhibition by CO and NO-- We recently identified an ODD domain within HIF-1alpha that plays a regulatory role for hypoxia-induced stabilization of HIF-1alpha . Internal removal of this domain rendered HIF-1alpha stable irrespective of oxygen tension (25). The inhibitory effect of CO and SNP on the accumulation of HIF-1alpha protein prompted us to examine whether the ODD domain is targeted by treatment with these agents. As in the experiment shown in Fig. 2, lanes 9-11 and 15-17, 293 cells were co-transfected with a vector expressing ARNT and another vector expressing wild-type HIF-1alpha or the ODD-deleted HIF-1alpha (401Delta 603; Fig. 3A). Consistent with previous results (25), normoxic cell extracts prepared from cells transfected with HIF-1alpha (401Delta 603) gave rise to strong constitutive HIF-1 binding (Fig. 3C, lane 4). In contrast to wild-type HIF-1alpha , HIF-1alpha (401Delta 603) was resistant to treatment with either CO (Fig. 3B, lane 4) or SNP (Fig. 3C, lane 6). These results indicate that the ODD domain is required for the suppressive effects of CO and SNP.


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Fig. 3.   The inhibitory effect of CO and NO is mediated through the ODD domain. A, schematic representation of three CMV-driven expression vectors: p(HA)HIF-1alpha , p(HA)HIF-1alpha (401Delta 603), and pGal4-ODD. 293 cells were transfected with p(HA)HIF-1alpha and pARNT (B, lanes 1 and 2; C, lanes 1-3), with p(HA)HIF-1alpha (401Delta 603) and pARNT (B, lanes 3 and 4; C, lanes 4-6), or with pGal4-ODD (C, lanes 7-9). Twenty-four hours after transfection, the cells were then subjected for 4 h to normoxia (N) or hypoxia (H) in the absence or presence of 80% CO (B) or 100 µM SNP (C). Cells extracts were then prepared and analyzed with EMSAs using 32P-labeled oligonucleotides containing an HIF-1 binding site (B, lanes 1-4; C, lanes 1-6) or Gal4 binding site (C, lanes 7-9). The endogenous HIF-1 complexes are marked with an arrowhead, whereas those composed of HIF-1alpha (401Delta 603) and ARNT are marked with asterisks, and Gal4 binding is marked with an arrow. PAS, periodic aryl hydrocarbon receptor-simultaneous.

Gal4 is normally a highly stable protein. However, when fused to the ODD domain, it undergoes oxygen-dependent degradation (25). To confirm that abrogation of HIF-1alpha abundance by SNP is specifically mediated by the ODD domain, a plasmid expressing Gal4-ODD was transfected into 293 cells and assayed for Gal4 DNA binding activity. Like wild-type HIF-1, Gal4-ODD exhibited very weak binding activity under normoxia (Fig. 3C, lane 7) and robust induction with hypoxia (Fig. 3C, lane 8). Of note, hypoxia-induced binding was markedly reduced by SNP treatment (Fig. 3C, lane 9).

In an effort to elucidate further the mechanism underlying the effect of nitric oxide on the ODD domain, we tested the possibility that NO could be forming an SNO adduct with a cysteine sulfhydryl group on the ODD. Accordingly, Cys-520, the only cysteine residue present in the ODD domain, was mutated to serine. As in experiments depicted in Figs. 2 and 3, HIF-1 binding activity was analyzed after cells were transfected with ARNT and either wild-type HIF-1alpha or the mutant HIF-1alpha (C520S). Interestingly, the mutant HIF-1alpha was still sensitive to SNP treatment (Fig. 4A, lane 3), which is in agreement with the loss of HIF-1alpha protein accumulation demonstrated by Western blotting (Fig. 4B, top panel). As shown in Fig. 4B, bottom panel, probing of the same blot with anti-ARNT antibodies demonstrated that ARNT protein levels remained unchanged.


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Fig. 4.   Cysteine nitrosylation of the ODD domain is not responsible for the effect of SNP. A, 293 cells were transfected with p(HA)HIF-1alpha (C520S) and pARNT (lanes 1-3) or p(HA)HIF-1alpha and pARNT (lanes 4-6), and were subjected to the same treatment as described in the legend to Fig. 3. EMSAs were performed with 32P-labeled oligonucleotides containing an HIF-1 binding site. B, the above cell extracts were also subjected to Western blot analyses with monoclonal anti-HA antibody (top panel) and polyclonal anti-ARNT antibody. HIF-1, induced binding; C, constitutive binding; N, nonspecific binding; F, free probe.

CO and NO Inactivate HIF-1alpha Transactivating Activity-- Previously Sogawa et al. (32) showed that SNP specifically inhibited hypoxia-induced activity of a luciferase reporter containing four copies of the HIF-1 binding site. Because we found that expression of HIF-1alpha (401Delta 603) gave rise to stable HIF-1 DNA binding activity that was insensitive to CO and SNP treatment (Fig. 3, B and C), we wondered whether this stable HIF-1 activity could transactivate an HIF-1 target reporter gene in the presence of SNP. A luciferase reporter EpoE-luc was used to test the effect of SNP in Hep3B cells co-transfected with wild-type HIF-1 (HIF-1alpha and ARNT) or the ODD domain-deleted HIF-1 (HIF-1alpha (401Delta 603) and ARNT) as mentioned above. The reporter EpoE-luc contains a native HIF-1 binding site within a 50-bp region of the human erythropoietin 3' enhancer (19). In agreement with the results of Sogawa et al. (32), SNP drastically inhibited hypoxia-induced luciferase activity when wild-type HIF-1 was transfected (Fig. 5, left). Surprisingly, a similar result was obtained when HIF-1alpha (401Delta 603) was tested (Fig. 5, right). It is noteworthy that HIF-1alpha contains two transactivation domains; one is within the ODD domain, and the other is at the C terminus (23, 24), in contrast to HIF-1alpha (401Delta 603), which has only the latter. Under normoxic conditions the transactivating activity of HIF-1alpha (401Delta 603) was substantially higher than that of the wild type, as observed previously (25). In addition, hypoxia further stimulated the reporter activity.


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Fig. 5.   Stabilized HIF-1alpha fails to transactivate HIF-1 target reporter in the presence of SNP. Reporter pEpoE-luc was co-transfected into Hep3B cells with p(HA)HIF-1alpha and pARNT (left) or p(HA)-HIF-1alpha (401Delta 603) and pARNT (right). Approximately 16-18 h before harvest, cells were incubated under normoxic or hypoxic conditions in the presence or absence of 100 µM SNP as indicated. Luciferase activities were measured as described (25), and the mean and S.E.s were plotted from three independent experiments.

These results raised the possibility that, in addition to impacting on the protein stability of HIF-1alpha , CO and NO may also affect its C-terminal activation domain. To address this question directly, we took advantage of a Gal4 fusion system in which the stable C-terminal transactivation domain was fused to the Gal4 DNA binding domain (Gal4-CAD, Fig. 6A) so that transactivating activity of the fusion protein could be assessed by a luciferase reporter containing five copies of the Gal4 binding site. Transactivating activity of the C-terminal portion of HIF-1alpha in a Gal4 fusion system can be enhanced by hypoxia without altering the protein abundance (23, 24). To that end, Hep3B cells were co-transfected with a reporter (Gal4-luc) and a vector expressing Gal4 alone (Gal4) or Gal4 fusion (Gal4-CAD) and subjected to normoxic or hypoxic treatment in the presence or absence of either CO or SNP. As expected, Gal4 alone failed to stimulate reporter activity when challenged by hypoxia (Fig. 7, B and C). In contrast, the reporter activity was markedly increased under hypoxia when the Gal4 fusion protein was expressed, but this induction was suppressed by both CO (Fig. 7B) and SNP (Fig. 7C). Taken together, these data indicate that CO and NO exerted their inhibitory effects by targeting the C-terminal transactivating domain of HIF-1alpha as well as its protein stability.


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Fig. 6.   CO and SNP specifically inhibit hypoxia-induced HIF-1alpha transactivation. A, schematic drawings of HIF-1alpha and Gal4-CAD. Reporter pGal4-luc was co-transfected into Hep3B cells with plasmids expressing Gal4 alone (left) or Gal4-CAD (right) and subjected to either normoxia (N) or hypoxia (H) for 16-18 h. Half of these cell culture plates were also treated with either 10% CO (B) or 100 µM SNP (C). B, mean luciferase activities of duplicate experiments; C, mean and S.E.s of luciferase activities from three experiments. PAS, periodic aryl hydrocarbon receptor-simultaneous.


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Fig. 7.   The effect of SNP is independent of the soluble guanylate cyclase pathway. Hep3B cells were incubated under normoxic (N, lane 1) or hypoxic (H, lanes 2 and 3) conditions for 4 h in the absence (lane 2) or presence (lane 3) of 1 mM 8-bromo-cGMP. HIF-1 DNA-binding was determined with EMSA. HIF-1, induced binding; C, constitutive binding; N, nonspecific binding; F, free probe.

NO Suppression Is Not Mediated by Guanylate Cyclase-- Because the predominant physiological action of nitric oxide is mediated through an increase in cGMP by activation of soluble guanylate cyclase (40), we also examined whether cell membrane-permeable 8-bromo-cGMP, an analog of cGMP, simulates the effect of SNP. No loss of HIF-1 binding was detected with up to 1 mM 8-bromo-cGMP (Fig. 7, lane 3). This result makes it very unlikely that the inhibitory effect of SNP is mediated through a pathway involving soluble guanylate cyclase.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have compared the effects of carbon monoxide and nitric oxide on the sensing of oxygen and the signaling pathway leading to HIF-1 activation. Although CO and NO both bind to ferrous iron in heme proteins, the two ligands differ in major respects. CO is chemically inert and undergoes no known chemical modifications in biological systems other than perhaps oxidation to CO2 coupled with reduction of cytochrome c oxidase (41) and other heme proteins (42). In contrast to CO, the biochemistry of NO is much more complex. NO binds to and activates its primary biological target, soluble guanylate cyclase, much more readily than does CO (43). Unlike CO, NO can also bind to ferric heme. Moreover, unlike CO, NO is capable of reacting with oxygen and superoxide (44), possibly affecting the signaling pathway for HIF-1 activation. Finally, unlike CO, NO can form S-nitroso derivatives of a broad repertoire of membrane, cytosolic, and nuclear proteins (44).

Our experiments show that CO and NO have similar effects on oxygen sensing and signaling: they both suppress expression of target genes through inhibition of HIF-1 activation, and both trigger destabilization of HIF-1alpha via the ODD domain and inhibition of the C-terminal activation domain. The most parsimonious mechanism is binding of these ligands to the heme group of the oxygen sensor. Our experiments with CO, along with those of Liu et al. (29), are at odds with a recent report by Srinivas et al. (45), who concluded that CO was not effective in suppressing expression of a hypoxia-inducible reporter gene. They tested only a single concentration of CO, 6%, and in fact observed ~17% suppression, which was statistically significant. We show in this report that the oxygen sensor has a low affinity for CO. Thus, it is likely that Srinivas et al. (45) would have obtained a more robust effect had they used a higher concentration of CO. Our finding that CO abrogates hypoxic stabilization of HIF-1alpha protein, in a dose-dependent manner (Fig. 2D), is in conflict with a recent report by Liu et al. (29), who found that 5% CO suppressed HIF-1 activation but did not affect the level of HIF-1alpha protein. In contrast to their experiments, we used the same cell extract for both EMSA and HIF-1alpha Western blot analysis. Alternatively, the discrepancy between our results and those of Liu et al. (29) may be explained in part by their use of only a single low concentration of CO.

Heme proteins vary markedly in their relative affinities for CO versus O2. Human hemoglobin binds CO ~210 times more tightly than O2. In contrast, the yeast flavohemoglobin binds CO and O2 with approximately equal affinity.2 Likewise, b-type cytochromes, which have been proposed to function in oxygen sensing (1, 38), are likely to have very low affinity for CO. We estimate that the oxygen sensor in Hep3B cells has a Haldane coefficient of ~0.5, a value significantly different from the coefficient of ~10 that Warburg (46) obtained for yeast cytochrome oxidase, which pertains to a wide range of higher organisms. This difference is difficult to reconcile with a recently proposed mitochondrial model of oxygen sensing (47, 48). Despite the inherent inaccuracies in our measurement of a "functional" Haldane coefficient for oxygen sensing, the value we have obtained may prove useful in screening candidate genes suspected to encode the oxygen sensor. An agreement between the "functional" value that we have obtained and a coefficient obtained from spectra of overexpressed protein would lend support to a candidate, whereas a significant discrepancy would rule it out.

The low affinity of the oxygen sensor for CO may have adaptive significance. The primary toxicity of CO in higher organisms is attributable to its high affinity binding to hemoglobin, locking the tetramer in the "oxy" conformation and thereby increasing oxygen affinity and decreasing oxygen unloading, resulting in tissue hypoxia. The organism needs intact oxygen sensors to adapt to this hypoxic stress. These sensors would be unresponsive to CO-induced hypoxia if they had high affinity for CO.

Our conclusion that CO and NO are acting at the same site is supported by experiments that rule out other known effects of NO. The fact that a mutation of the single cysteine in the ODD domain did not affect the function of HIF-1 (Fig. 4) implies that the NO effect is not attributable to S-nitrosylation of HIF-1alpha . Moreover, the fact that the C520S mutation has no effect on the activation of HIF-1 and its suppression by NO argues that SH-dependent redox chemistry is not involved in oxidant-dependent degradation. This result contrasts strikingly with an analogous phenomenon, the iron-dependent degradation of iron regulatory protein 2, the protein that regulates either the translation or the stability of mRNAs encoding proteins critical in iron homeostasis. Iwai et al. (49) have recently shown that a cluster of conserved cysteine residues is required for the iron-dependent oxidative attack on the protein.

An alternate explanation for the suppressive effect of NO is via binding to its most prominant target, guanylate cyclase. In contrast to findings of Liu et al. (29) but similar to those reported by Sogawa et al. (32), we found no effect of 8-bromo-cGMP on HIF-1 activation (Fig. 5), making it unlikely that suppression of HIF-1 activation depends on up-regulation of guanylate cyclase.

It is possible that NO and CO act independently of one another with NO generating reactive oxygen species (ROS) directly, whereas CO could generate ROS indirectly through redox cycling of cytochrome c (46) and other electron acceptors (42), including the oxygen sensor. Cytochrome c participates in the oxidation of CO to CO2 by reduction of its heme iron. Oxidation of cytochrome c back to the ferric state would reduce oxygen to superoxide, enabling the generation of ROS via the iron-catalyzed Fenton reaction.

Our work supports and extends the paradigm that HIF-1 activation correlates with the abundance of HIF-1alpha protein. In addition, both CO and NO suppressed hypoxic induction of the C-terminal activation domain, which is independent of HIF-1alpha stability. Our demonstration of the inhibitory effect of CO and NO on HIF1alpha stability and transactivation supports the notion of two distinct pathways that lead to inhibition of HIF-1 activity. The conjoint destabilization of HIF-1alpha and inhibition of C-terminal transactivation of HIF-1alpha are both distal events in the signaling pathway, triggered by CO and NO, which act appear to act proximally, very likely at the level of the oxygen sensor. The only difference between the two ligands that we could find was that the induction of Epo mRNA by cobalt was suppressed by NO but not by CO (Fig. 1). This result is consistent with our hypothesis that this transition metal can substitute for iron in the oxygen sensor and mimic deoxy heme (27). Carbon monoxide cannot bind to cobalt-substituted heme, whereas NO binds to cobalt heme with high affinity (50). However, the action of cobalt may be independent of the oxygen sensor, as others have suggested (48, 51). In contrast to cobalt, the activation of HIF-1 by the iron chelator desferrioxamine is unlikely to directly affect oxygen sensing, consistent with the inability of CO to suppress induction by this agent. The most plausible explanation of the effect of desferrioxamine is that free iron functions as a Fenton reagent, catalytically increasing levels of ROS. Therefore iron chelation will result in a lower level of ROS, which appears to be critical in signaling the activation of HIF-1 (1).

    ACKNOWLEDGEMENTS

We thank Shoumo Bhattacharya, Zoltan Arany, and David M. Livingston for providing pGal4-luc, pSB92, and anti-HIF-1alpha antibodies, Patrick Maxwell for providing anti-HIF-1alpha antibody, and Maureen Schau for technical assistance.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grants RO1-DK41234 (to H. F. B.) and RO1-DK45098 (to M. A. G.) and by National Institutes of Health Individual National Research Service Award F32-DK09365 (to L. E. H.).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.

Dagger Contributed equally to this work.

§ To whom correspondence should be addressed: Hematology-Oncology Division, LMRC-2, Brigham & Women's Hospital, Harvard Medical School, 221 Longwood Ave., Boston, MA 02115. Tel.: 617-732-5841; Fax: 617-739-0748; E-mail: bunn{at}calvin.bwh.harvard.edu.

2 H. Zhu, J. Olsen, and A. Riggs, personal communicacation.

    ABBREVIATIONS

The abbreviations used are: HIF-1, hypoxia-inducible factor 1; ODD, oxygen-dependent degradation; SNP, sodium nitroprusside; Epo, erythropoietin; CMV, cytomegalovirus; HA, hemagglutinin; EMSA, electrophoretic mobility shift assay; luc, luciferase; ROS, reactive oxygen species; ARNT, aryl hydrocarbon nuclear translocater.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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