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Nitric Oxide Donor, (±)-S-Nitroso-N-acetylpenicillamine, Stabilizes Transactive Hypoxia-Inducible Factor-1 by Inhibiting von Hippel-Lindau Recruitment and Asparagine Hydroxylation

Department of Life Science, University of Seoul, Seoul, Korea (Y.-K.P., M.O., T.L., H.P.); Life Science Division, Korea Institute of Science and Technology, Seoul, Korea (D.-R.A., E.G.Y.); and Research Labs of Dong-A Pharm. Co. Ltd., Kyunggi-do, Korea (M.S.)

Received January 14, 2008; accepted April 21, 2008

	Abstract

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We have confirmed that the NO donor (±)-S-nitroso-N-acetylpenicillamine (SNAP) stabilizes the transactive form of hypoxia-inducible factor-1 (HIF-1), leading to the induction of HIF-1 target genes such as vascular endothelial growth factor and carbonic anhydrase 9. Activation of HIF-1 should require inhibition of the dual system that keeps it inactive. One is ubiquitination, which is triggered by hydroxylation of HIF-1-proline and the subsequent binding of E3 ubiquitin ligase, the von Hippel Lindau (VHL) protein. The other is hydroxylation of HIF-1-asparagine, which reduces the affinity of HIF-1 for its coactivator, cAMP responsive element binding protein/p300. We examined the effects of the NO donor SNAP on proline and asparagine hydroxylation of HIF-1 peptides by measuring the activities of the corresponding enzymes, HIF-1-specific proline hydroxylase 2 (PHD2) and the HIF-1-specific asparagine hydroxylase, designated factor inhibiting HIF-1 (FIH-1), respectively. We found that the SNAP did not prevent PHD2 from hydroxylating the proline of HIF-1. Instead, it blocked the interaction between VHL and the proline-hydroxylated HIF-1, but only when the reducing agents Fe(II) and vitamin C were limiting. The fact that the absence of cysteine 520 of HIF-1 abolishes its responsiveness to SNAP suggests that this residue mediates the inhibition by SNAP of the interaction between VHL and HIF-1, presumably by S-nitrosylation of HIF-1. Un-like PHD2, asparagine hydroxylation by FIH-1 was directly inhibited by SNAP, but again only when reducing agents were limiting. Substitution of cysteine 800 of HIF-1 with alanine failed to reverse the inhibitory effects of SNAP on asparagine hydroxylation, implying that FIH-1, not its substrate HIF-1, is inhibited by SNAP.

View larger version (59K): [in this window] [in a new window]   Fig. 1. Effects of NO-related drugs on HIF-1 and its target genes. HeLa cells were treated with the NO donor drugs, NOC18, spermine NONOate, SNAP, and GSNO at different times and doses. A, the protein level of HIF-1 in each sample was visualized by Western blot analysis using anti-HIF-1 antibody (Choi et al., 2006b). The SDS-PAGE gels were stained with Coomassie blue to confirm equal loading. HIF-1 protein levels were quantified by measuring band intensities of HIF-1, of which HeLa cells were exposed to SNAP (200 µM), spermine NONOate (200 µM), or hypoxia for 4 h. B, the mRNA level of VEGF (top) was detected by Northern analysis and the mRNA level of carbonic anhydrase 9 (bottom) by RT-PCR. By using quantitative real-time PCR, we measured mRNAs of VEGF and CA9, of which HeLa cells were exposed to SNAP (200 µM), spermine NONOate (200 µM), or hypoxia for 6 h. C, HRE activity was measured by transfecting HeLa cells with the HRE-driven luciferase reporter plasmid p(HRE)4-luc (100 ng), together with a β-galactosidase encoding plasmid, pCHO110 (50 ng), into 1x105 HeLa cells. Luciferase activities were normalized by β-galactosidase activities. The values given are means and standard deviations of at least five experiments.

View larger version (49K): [in this window] [in a new window]   Fig. 2. Effect of SNAP on the ubiquitination of HIF-1. A, HeLa cells were exposed to SNAP, spermine NONOate, the proteasome inhibitor MG132, hypoxia, and HIF-1-ubiquitination blocker TPEN (Choi et al., 2006a) for 4 h under normoxic conditions. HIF-1 protein was detected by Western blot analysis. HIF-1 and ubiquitinated HIF-1 are indicated. N, normoxia; H, hypoxia (1% oxygen). B, 293 cells were transfected with Flag-HIF-1 and HA-ubiquitin as indicated. The transfected cells were treated with SNAP, MG132, or hypoxic mimicker CoCl2 for 4 h before harvest. Whole cell lysates (300 µg) were immunoprecipitated (IP) with anti-HA antibody and then probed using anti-Flag antibody by Western blot (WB) analysis. HSP70 was detected to confirm equal loading.

In the current study, we dissected the mechanism of activation of HIF-1 and tested the effects of the NO donor (±)-S-nitroso-N-acetylpenicillamine (SNAP) on each step. We found that SNAP did not block proline hydroxylation of HIF-1 by PHD2 but inhibited the interaction between VHL and hydroxylated HIF-1, leading to the accumulation of HIF-1. In contrast, SNAP directly inhibited the asparagine hydroxylation activity of FIH-1, so rescuing the interaction between HIF-1 and CBP.

	Materials and Methods

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Cells and Reagents. Human epithelial HeLa cells were cultured and exposed to hypoxia (1% O₂) as described previously (Choi et al., 2005). NO donors, N-(2-aminoethyl)-N-(2-hydroxy-2-nitrosohydrazino)-1,2-ethylenediamine (spermine NONOate), NOC18, and SNAP, and zinc chelator N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN) were purchased from Calbiochem (San Diego, CA), and all other chemicals were from Sigma Chemical (St. Louis, MO). The cDNAs used were HIF-1 (GenBank accession number U22431 [GenBank] ), FIH-1 (GenBank accession number AF395830 [GenBank] ), PHD2 (GenBank accession number AJ310543 [GenBank] ), and VHL (GenBank accession number AF010238 [GenBank] ) (Choi et al., 2005, 2006b).

Measurement of PHD Activity by VHL Pull-Down Assay. The catalytic domain (amino acids 184-418) of human PHD2 was cloned into the pET21b His2(+) vector, overexpressed in Escherichia coli as a histidine-tagged fusion protein, and purified by nickel-affinity chromatography (Choi et al., 2005). The in vitro VHL pull-down assay was performed as described previously (Jaakkola et al., 2001; Choi et al., 2005). In brief, [³⁵S]methionine-labeled VHL protein was synthesized by in vitro translation using the pcDNA3.1/hygro-VHL plasmid, according to the instruction manual (Promega, Madison, WI). GST-ODD (amino acids 401-603 of human HIF-1) was expressed in E. coli and purified with glutathione-resin (BD Biosciences). Resin-bound GST-ODD (2 µg of protein/50 µl of resin volume) was incubated in the presence of 5 mM -ketoglutarate with 1 to 2 µg of histidine-tagged PHD2 (184-418 amino acid) and other cofactors as indicated, in 200 µl of NETN buffer (20 mM Tris, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P40, and 1 mM phenylmethylsulfonyl fluoride) with mild agitation for 90 min at 30 °C. The reaction mixture was centrifuged and washed three times with 10 volumes of NETN buffer. Resin-bound GST-ODD was mixed with 10 µl of [³⁵S]-VHL in 500 µl of EBC buffer (50 mM Tris, pH 8.0, 120 mM NaCl, and 0.25% Nonidet P40). After mild agitation at 4 °C for 2 h, the resin was washed three times with 1 ml of NETN buffer, and proteins were eluted in 3x SDS sample buffer, fractionated by 12% SDS-PAGE, and detected by autoradiography. The amount of each sample loaded was monitored by staining the GST-ODD with Coomassie blue. Instead of GST-ODD, HIF-1 peptide was used as substrate. Biotinylated human HIF-1 (amino acids 556-575) peptide (mol. wt. 2637.0) (biotin-DLDLEMLAPYIPMDDDFQLR; 2 µg) was preincubated with 1 µg of his-PHD2 (amino acids 184-418) in a final volume of 100 µl in NETN buffer containing 5 mM -ketoglutarate with other cofactors as indicated at 30°C for 90 min. ImmunoPure immobilized monomeric avidin (Pierce Chemical) (30 µl of a 50% slurry) was preincubated with 3 mg of bovine serum albumin for 5 min at room temperature. The avidin was added to the above-mentioned hydroxylation reaction mixture, which was incubated with mild agitation for 60 min at 22°C. The avidin-associated peptide was washed three times with 1 ml of NETN buffer and then mixed with 10 µl of ³⁵S-labeled VHL in 500 µl of EBC buffer with mild agitation at 4°C for 1 h (Choi et al., 2005). The resin was washed four times with 1 ml of NETN buffer, and proteins were eluted, analyzed by 12% SDS-PAGE, and autoradio-graphed.

View larger version (37K): [in this window] [in a new window]   Fig. 3. Effects of SNAP on HIF-1-proline hydroxylation-dependent VHL recruitment. Resin-bound GST-HIF-1 (amino acids 401-603) was incubated with recombinant His-tagged PHD2 (amino acids 184-418) (1 µg) in the presence or absence of SNAP with 100 µM Fe(II), 2 mM vitamin C, and 5 mM -KG (A), with 2 mM vitamin C and 5 mM -KG (B); or with only 5 mM -KG (C). After the hydroxylation reaction, resin-bound GST-HIF-1 (amino acids 401-603) was washed and mixed with in vitro-translated 35S-labeled VHL. [35S]VHL capture by GST-HIF-1 (amino acids 401-603) was visualized by SDS-PAGE and autoradiography. Sample loading was monitored by measuring GST-HIF-1 (amino acids 401-603) protein stained with Coomassie blue. The first lane represents 10% of the [35S]VHL used. D, HeLa cells were exposed to either SNAP (200 µM) or hypoxia (1% O2) with or without vitamin C (100 µM) or FeCl2 (100 µM) for 4 h. HIF-1 was detected by Western blot analysis. HSP70 was detected by Western blot analysis for loading control.

Mass Spectrometric Analysis. After hydroxylation for 2 h at room temperature, excess salts were removed from substrate peptides with ZipTip_C18 (Millipore, Billerica, MA). The peptide was eluted from the tip with -cyano-4-hydroxycinnamic acid in acetoni-trile/water containing 0.1% trifluoroacetic acid [50:50 (v/v)] followed by extensive washing with 0.1% trifluoroacetic acid in water. The eluted peptide solution was transferred to a MALDI sample plate, and MALDI-TOF measurements were performed with a Voyager analyzer (Applied Biosystems, Foster City, CA).

Western Blot Analysis and Coimmunoprecipitation. HeLa cells were grown to 80% confluence on 100-mm tissue culture plates and treated with drugs or hypoxia for 4 h. Whole cell extracts were prepared as described previously (Choi et al., 2006b). For immunoprecipitation, 200 µg of whole cell lysates was incubated with 1 µg of anti-CBP antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at 4°C overnight. The resulting immunocomplexes were analyzed by Western blotting with anti-human HIF-1 antibody (BD Biosciences, San Jose, CA) (Choi et al., 2006b).

Transient Transfection and Luciferase Assay. Transfection of HRE-driven reporter plasmids (100 ng) and pCHO110 (50 ng), which encodes the β-galactosidase gene, into 1 x 10⁵ HeLa cells was carried out using Lipofectamine Plus reagent (Choi et al., 2006b). Because the transfected β-galactosidase gene is transcribed by a constitutive promoter, β-galactosidase activity can represent the transfection efficiency of each sample. The measurements of luciferase activity were normalized for β-galactosidase.

Northern Analysis and Reverse Transcriptase PCR Analysis. Total RNA (10 µg) was used for Northern blot analysis. Blots were hybridized with 25 ng of -³²P-labeled DNA fragments encoding vascular endothelial growth factor (VEGF) as described previously (Yim et al., 2003). Reverse-transcription PCR (RT-PCR) was performed with total RNA (1 µg), and 1 µM concentration of each primer of carbonic anhydrase-9 (CA9) (sense, 5'-CTGTCACTGCTGCTTCTGAT-3'; antisense, 5'-TCCTCTCCAGGTAGATCCTC-3') (Choi et al., 2006b). The PCR products were analyzed on ethidium bromide-stained agarose gels.

Quantitative Real-Time RT-PCR. cDNA was quantified by real-time PCR on the iQTM SYBR Green Supermix and MyiQ single color real-time PCR detection system (Bio-Rad, Hercules, CA) were used. We used the following primers: human VEGF: forward, 5'-AACCATGAACTTTCTGCTGTCTTG-3'; reverse, 5'-TTCACCACTT-CGTGATGATTCTG-3'; human CA9: forward, 5'-CAGTTGCTGTCTCGCTTGGA-3'; reverse, 5'-TGAAGTCAGAGGGCAGGAGTG-3'; and 18S, forward, 5'-ACCGCAGCTAGGAATAATGGAATA-3'; reverse, 5'-CTTTCGCTCTGGTCCGTCTT-3'. The expression level of 18S rRNA was used for normalization. All PCRs were performed in triplicate. We present the average and standard deviation of at least three experiments.

View larger version (33K): [in this window] [in a new window]   Fig. 4. Effect of SNAP on proline-hydroxylation of HIF-1.A,2 µM biotin-HIF-1-(556-575) peptide (mol. wt. 2637.0) and 100 µM -KG were incubated together with the following combinations of the three reagents: 1 µg of recombinant His-PHD2 (amino acids 184-418), 400 µM vitamin C, and SNAP as indicated; Fe(II) was not included. The reaction mixtures were analyzed by MALDI-TOF as described previously (Cho et al., 2007). The detected mass of the major peptide was shown above the peak. B, 2 µg of biotin-HIF-1 (amino acids 556-575) peptide (mol. wt. 2637.0) was hydroxylated in the absence or presence of SNAP by adding recombinant His-PHD2 (amino acids 184-418) (1 µg) and 400 µM -KG. Biotin-HIF-1 (amino acids 556-575) peptide was then isolated using immobilized avidin-resin and mixed with labeled VHL (Choi et al., 2005). [35S]VHL capture by hydroxylated biotin-HIF-1 (amino acids 556-575) peptide was visualized by SDS-PAGE and autoradiography. The first lane represents 10% of the [35S]VHL used.

	Results

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SNAP Blocks VHL Binding of HIF-1. To test whether SNAP acts by blocking ubiquitination, we examined the effect of the protease inhibitor MG132. Western blot analysis showed that treatment with MG132 stabilized the ubiquitinated high molecular weight form of HIF-1 in normoxic conditions. Zinc chelator TPEN specifically inhibits the ubiquitination of HIF-1 (Choi et al., 2006a). Like TPEN, SNAP and spermine NONOate reduce the high molecular weighted HIF-1 and indeed inhibit ubiquitination (Fig. 2). We co-transfected HA-tagged ubiquitin and Flag-tagged HIF-1 and then performed coimmunoprecipitation assay by using anti-HA antibody. We confirmed that SNAP reduced the ubiquitinated form of HIF-1 (Fig. 2B). O₂-dependent ubiquitination of HIF-1 is mediated by an HIF-1-specific E3 ligase named VHL. Because VHL specifically recognizes and binds to the hydroxylated proline residues of the ODD domain (amino acids 401-603), HIF-1-specific proline hydroxylation is the initial event in HIF-1 degradation. To assess the effect of SNAP on the hydroxylation and VHL binding ability of HIF-1, we used bacterially expressed and purified GST-HIF-1 (amino acids 401-603) fusion protein as substrate. Because we used the glutathione resin-bound form of GST-HIF-1 (amino acids 401-603), glutathione was not present in the subsequent reactions. GST-HIF-1 (amino acids 401-603) was incubated with the purified recombinant histidine-PHD2 (amino acids 184-418) fusion protein, which contains the catalytic domain, in the presence of -ketoglutarate, Fe (II), and vitamin C (Choi et al., 2005). The addition of PHD2 increased capture of VHL protein, indicating that it hydroxylates the proline residues of HIF-1 (Fig. 3A). We added SNAP to the resin-bound GST-HIF-1 together with PHD2 and cofactors as indicated (Fig. 3A). The presence of SNAP (100 and 500 µM) in the hydroxylation reaction did not significantly inhibit the recruitment of VHL by HIF-1.

View larger version (35K): [in this window] [in a new window]   Fig. 5. Effect of SNAP and cysteine 520 of HIF-1. A, resin-bound GST-HIF-1 (amino acids 401-603) was incubated with his-PHD2 (amino acids 184-418) (1 µg) and 5 mM -KG in the presence or absence SNAP (200 or 500 µM) for 90 min at 30°C. Then the resin-bound GST-HIF-1 (amino acids 401-603) was washed and mixed with in vitro translated 35S-labeled VHL. [35S]-VHL capture by GST-HIF-1(401-603) was visualized by SDS-PAGE and autoradiography. Sample loading was monitored by measuring GST-HIF-1 (amino acids 401-603) protein stained with Coomassie blue. The first lane represents 10% of the [35S]VHL used. B, resin-bound GST-HIF-1 (amino acids 401-603) C520A mutant protein was used instead of GST-HIF-1 (amino acids 401-603).

SNAP Fails to Inhibit Proline Hydroxylation. To determine whether SNAP inhibits the hydroxylation reaction or VHL recruitment, we directly assessed its effect on hydroxylation of Pro564 of HIF-1 by measuring changes in the molecular weight of HIF-1 peptide (amino acids 556-575) by mass spectroscopy. For the hydroxylation reaction we used purified His-tagged PHD2 (amino acids 184-418), and -ketoglutarate, in the absence of Fe(II). After incubation with PHD2, the peptide yielded a new MALDI-TOF peak corresponding to an increase of molecular weight of 16, both in the presence and the absence of vitamin C (Fig. 4A). The molecular weight also increased in the presence of SNAP, indicating that SNAP stabilizes HIF-1 by inhibiting the interaction between VHL and hydroxylated HIF-1 rather than by inhibiting the proline-hydroxylation reaction. To confirm this, we used the HIF-1 peptide (amino acids 556-575) in the hydroxylation-dependent VHL pull-down assay instead of GST-HIF-1 (amino acids 401-603). The ODD domain of HIF-1 contains one cysteine residue (residue 520), but the HIF-1 peptide contains no cysteine. PHD2 increased the capture of VHL protein, indicating that it hydroxylates the peptide. However, SNAP failed to inhibit VHL recruitment by the HIF-1 peptide even in the absence of Fe(II) and vitamin C, suggesting that SNAP inhibition requires the cysteine residue of HIF-1 (Fig. 4B). As a control experiment, we added the purified GST protein in the HIF-1 peptide (556-575)/VHL pull-down assay. The results showed that GST alone had no effect on association of HIF-1 peptide (amino acids 556-575) with VHL in the presence of SNAP, confirming that the cysteine residue of HIF-1-ODD but not of GST mediates the effect of SNAP (supplemental data).

Involvement of Cysteine 520 of HIF-1. To test whether S-nitrosylation is involved, we created a point mutation substituting alanine for cysteine 520. The result shown in Fig. 5 indicates that replacement of the cysteine abolishes the response of HIF-1 to SNAP in the hydroxylation-dependent VHL pull-down assay, implying that S-nitrosylation of Cys520 blocks the interaction between hydroxylated HIF-1 and VHL. Our results indicate that SNAP stabilizes HIF-1 by blocking VHL recruitment not by inhibiting proline hydroxylation. The S-nitrosylation of Cys520 presumably prevents hydroxylated HIF-1 from recruiting VHL.

Effects of SNAP on Asparagine Hydroxylation and CBP Binding of HIF-1. To induce transactivation capability, the stabilized HIF-1 needs to recruit a coactivator, CBP/p300. In normoxic conditions, the asparagine 803 is hydroxylated by FIH-1, and this interferes with the interaction between CBP/p300 and HIF-1. Therefore, FIH-1 activity is inversely related to the recruitment of CBP/p300. To test the effect of SNAP on FIH-1 activity, we incubated the HIF-1 peptide (amino acids 788-822) with purified recombinant His-tagged FIH-1 in the presence and absence of SNAP. After treatment with FIH-1, -ketoglutarate (-KG), and a minimal amount of vitamin C (2 µM) in the absence of Fe(II), the peptide gave the characteristic new MALDI-TOF peak (Fig. 6A) (Cho et al., 2007). SNAP prevented FIH-1 from hydroxylating the peptide (Fig. 6A), whereas its effect was abolished in the presence of excess vitamin C (400 µM) (Fig.6B). Thus the asparagine-hydroxylating activity of FIH-1 is only inhibited by SNAP when reducing agents are limiting.

View larger version (27K): [in this window] [in a new window]   Fig. 6. Effects of NO donors on asparagine hydroxylation of HIF-1. A, FITC-HIF-1 (amino acids 788-822) peptide (mol. wt. 4332.2) (3 µM), -KG (100 µM), and vitamin C(2 µM) were mixed with His-tagged full-length FIH-1 (1 µg) in hydroxylation buffer in the presence or absence of SNAP (200 or 500 µM). B, FITC-HIF-1 (amino acids 788-822) peptide (3 µM), -KG (100 µM), and vitamin C (400 µM) were mixed with His-FIH-1 (1 µg) in the presence or absence of SNAP (500 µM). Fe(II) was not added. The mixtures were incubated at 30°C for 2 h, followed by MALDI-TOF analysis. Note that the indicated molecular masses correspond to the peptides with detached FITC (mol wt. 389.0) in a sodium (mol. wt. 23.0)-added form (mol. wt. 3965.7) during the MALDI-TOF measurements. The detected mass of the major peptide is shown above the peak.

HIF-1 peptide (amino acids 786-826) contains one cysteine. To test whether the inhibitory effect of SNAP on FIH-1 activity depends on this residue, we used the alanine-substituted mutant peptide (amino acids 786-826). The mass analysis in Fig. 7A shows that FIH-1 was still able to increase the molecular weight of the mutant HIF-1 peptide and that SNAP also inhibited its hydroxylation. The fact that cysteine 800 is not essential for the inhibitory effect of SNAP on the asparagine hydroxylation of HIF-1 implies that SNAP does not inhibit FIH-1 activity by S-nitrosylation of this residue.

View larger version (35K): [in this window] [in a new window]   Fig. 7. The effect of SNAP and cysteine 800 of HIF-1. A, in the presence or absence of SNAP (500 µM), 3 µM FITC-HIF-1 (amino acids 788-822) peptide (top) or 3 µM FITC-HIF-1 (amino acids 786-826) C800A peptide (mol. wt. 4975.3) (bottom) was incubated along with His-FIH-1 (1 µg) with -KG (100 µM) and vitamin C (2 µM) at 30°C for 2 h followed by MALDI-TOF analysis (Cho et al., 2007). Note that the indicated molecular weights correspond to the peptides with detached FITC (mol. wt. 389.0) in a sodium (mol. wt. 23.0)-added form (mol. wt. 4608.8) during the MALDI-TOF measurements. The detected mass of the major peptide is shown above the peak. B, effects of SNAP and spermine NONOate on CBP recruitment by HIF-1 in vivo. HeLa cells were exposed to SNAP (200 µM), spermine NONOate (200 µM), or hypoxic conditions (1% oxy-gen) for 4 h. Whole cell lysates were immunoprecipitated with anti-CBP antibody, and the resulting immunocomplexes were analyzed using anti-human HIF-1 and anti-CBP. To confirm equal loading, HSP70 was detected with anti-HSP70 antibody.

	Discussion

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We have confirmed that SNAP stabilizes the transactive form of HIF-1, leading to the induction of HIF-1 target genes such as VEGF and CA9. We examined the direct effects of NO donors on the hydroxylation of HIF-1 peptides by PHD2 and FIH-1 in vitro. The finding that SNAP reduced the high molecular weight form of HIF-1 indicated that it inhibits the ubiquitination of HIF-1, thereby stabilizing it. SNAP treatment failed to stabilize cellular HIF-1 when the cells were cotreated with vitamin C, indicating that cellular redox status affects SNAP action, presumably NO release from SNAP. Our observations imply that FIH-1, but not its substrate HIF-1, is directly inhibited by SNAP. In agreement with this, the coactivator CBP was able to interact with HIF-1 stabilized by SNAP in the cellular context.

It has been reported that the NO donors GSNO and SNAP inhibit the proline hydroxylation-dependent VHL recruitment (Metzen et al., 2003). In contrast, others found that NO donors, including SNP, PAPA NONOate, and MAHMA NONOate, rather increase the proline hydroxylation-dependent VHL interaction (Wang et al., 2002). However, Li et al. (2007) demonstrated recently, using an antibody directed against the hydroxylated proline of HIF-1, that in GSNO-treated mouse 4T tumor cells, proline-hydroxylated HIF-1 remained detectible. They also demonstrated by a biotin switch assay, that Cys533 of mouse HIF-1 (equivalent to Cys520 of human HIF-1) is nitrosylated, and that this S-nitrosylation does not inhibit proline hydroxylation. By using hydroxylation-dependent VHL pulldown assay and mass analysis, we confirmed here that SNAP blocks VHL recruitment but not proline hydroxylation of HIF-1, and that this inhibitory effect is reversed by reducing agents such as vitamin C and Fe(II).

View larger version (19K): [in this window] [in a new window]   Fig. 8. Schematic diagram of the effects of SNAP on the HIF-1 regulation. For details, see Discussion.

The importance of HIF-1 has been emphasized in the context of therapeutic interventions in many diseases, especially cancer and recently inflammation. The finding that the catalytic activities of both PHDs and FIH-1 require vitamin C, Fe(II), -ketoglutarate, and molecular oxygen provides insight into how HIF-1 can be activated by changes in mitochondrial activity, ROS or the pool of reducing agents including NADH, vitamin C, and glutathione. The inhibitory effect of antioxidants including vitamin C (Lu et al., 2005; Gao et al., 2007) and Fe(II) (Gerald et al., 2004; Lu et al., 2005) on tumorigenesis has been re-evaluated in the context of HIF-1 destabilization. Gao et al. (2007) reported that antioxidants such as N-acetylcysteine and vitamin C inhibit lymphoma xenografts by diminishing HIF-1 levels in a prolyl hydroxylase 2- and von Hippel-Lindau protein-dependent manner (Gao et al., 2007). In addition, trichloroacetic acid cycle intermediates are involved in the HIF-1 hydroxylation reaction. For PHDs, succinate as a product is a competitive inhibitor of -ketoglutarate, the substrate (Selak et al., 2005; Pan et al., 2007). Fumarate and malate are other trichloroacetic acid cycle intermediates shown to negatively regulate PHD (Isaacs et al., 2005; Pan et al., 2007) but not FIH activity (Hewitson et al., 2007; Koivunen et al., 2007). In addition, mitochondrial ROS are essential for proper O₂ sensing by PHDs. In cytochrome c null embryonic cells, which have defects in generating mitochondrial ROS, HIF- protein remains hydroxylated by PHDs and so cannot be stabilized even in moderate hypoxia (1.5% O₂ for 4 h) (Mansfield et al., 2005), whereas exogenous treatment with H₂O₂ restores HIF- stabilization even in the absence of cytochrome c.

Although PHDs and FIH-1 share common cofactors, the catalytic domains of both enzymes are different. FIH-1 has jumonji domain, which differs from the catalytic domain of PHD2. These two hydroxylation enzymes respond differently to inhibitors. Succinate and fumarate inhibit PHD but not FIH-1 activity (Hewitson et al., 2007; Koivunen et al., 2007). Desferrioxamine and several metals are effective inhibitors of FIH-1 but ineffective inhibitors of PHDs in vitro (Hirsiläet al., 2005). Here, we demonstrate that SNAP inhibits FIH-1 but not PHD2. Instead, it inhibits VHL recruitment, stabilizing the transactive form of HIF-1 in normoxic cells. NO is released in many pathophysiological conditions, including inflammation and blood clotting (Vadseth et al., 2004; Li et al., 2007; Yeo et al., 2008). In these conditions, a variety of mediators may interfere with the NO effects on the regulation of HIF-1. It remains to be seen whether the endogenous NO also has dual inhibitory effects on VHL recruitment and asparagine hydroxylation, leading to functional HIF-1, and whether the NO effects can be modulated by other inflammatory mediators (Li et al., 2007).

	Footnotes

 
This study was supported by grant 2004-01969 from the Neurobiology Research Program, grant R01-2007-000-11672-0 from the Korea Science and Engineering Foundation, by grant CBM1-B113-001-1-0-0 from the center for Biological Modulators of the 21st Century Frontier R&D Program of the Ministry of Science and Technology (to H.P.), and by grant from the Korea Institute of Science and Technology (to E.G.Y.). Y.-K.P. was supported by a Brain Korea 21 Research Fellowship from the Ministry of Education and Human Resources Development and by a Seoul Science Fellowship.

All experiments reported in this article were repeated with similar results.

ABBREVIATIONS: HIF-1, hypoxia-inducible factor-1; -KG, -ketoglutarate; CA9, carbonic anhydrase 9; CBP, cAMP-responsive element binding protein; FIH-1, factor inhibiting hypoxia-inducible factor-1; HRE, hypoxia-responsive element; ODD, oxygen-dependent degradation domain of hypoxia-inducible factor-1; PHD, hypoxia-inducible factor-1-specific prolyl hydroxylase; ROS, reactive oxygen species; SNAP, (±)-S-nitroso-N-acetylpenicillamine; spermine NONOate, N-(2-aminoethyl)-N-(2-hydroxy-2-nitrosohydrazino)-1,2-ethylenediamine; TPEN, N,N, N',N'-tetrakis (2-pyridylmethyl) ethylenediamine; VHL, von Hippel-Lindau; VEGF, vascular endothelial growth factor; HSP70, 70-kDa heat shock protein; FITC, fluorescein isothiocyanate; PAGE, polyacrylamide gel electrophoresis; NOC18, 2,2'-(hydroxynitrosohydrazino) bis-ethanamine; GSNO, S-nitrosoglutathione; MALDI-TOF, matrix-assisted laser desorption ionization/time of flight; PCR, polymerase chain reaction; RT-PCR, reverse transcriptase polymerase chain reaction; HA, hemagglutinin; MG132, N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal.

The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.

Address correspondence to: Dr. Hyunsung Park, 90 Cheonnong-dong, Tongdaemun-gu, Seoul, 130-743, Republic of Korea. E-mail: hspark{at}uos.ac.kr

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