Nitric Oxide Induces Hypoxia-inducible Factor 1 Activation That Is Dependent on MAPK and Phosphatidylinositol 3-Kinase Signaling*

Hypoxia-inducible factor-1 (HIF-1) is a master regulator of cellular adaptive responses to hypoxia. Levels of the HIF-1α subunit increase under hypoxic conditions. Exposure of cells to certain nitric oxide (NO) donors also induces HIF-1α expression under nonhypoxic conditions. We demonstrate that exposure of cells to the NO donor NOC18 or S-nitrosoglutathione induces HIF-1α expression and transcriptional activity. In contrast to hypoxia, NOC18 did not inhibit HIF-1α hydroxylation, ubiquitination, and degradation, indicating an effect on HIF-1α protein synthesis that was confirmed by pulse labeling studies. NOC18 stimulation of HIF-1α protein and HIF-1-dependent gene expression was blocked by treating cells with an inhibitor of the phosphatidylinositol 3-kinase or MAPK-signaling pathway. These inhibitors also blocked NOC18-induced phosphorylation of the translational regulatory proteins 4E-BP1, p70 S6 kinase, and eIF-4E, thus providing a mechanism for the modulation of HIF-1α protein synthesis. In addition, expression of a dominant-negative form of Ras significantly suppressed HIF-1 activation by NOC18. We conclude that the NO donor NOC18 induces HIF-1α synthesis under conditions of NO formation during normoxia and that hydroxylation of HIF-1α is not regulated by NOC18.

Hypoxia induces a series of adaptive physiological responses (1). At the cellular level, the adaptation involves a switch of energy metabolism from oxidative phosphorylation to anaerobic glycolysis, increased glucose uptake, and the expression of stress proteins related to cell survival or death (2). At the molecular level, the adaptation involves changes in mRNA transcription and mRNA stability (2,3). One of the most important transcription factors that activates the expression of oxygen-regulated genes including vascular endothelial growth factor (VEGF) 1 and inducible nitric-oxide synthase is hypoxia-inducible factor 1 (HIF-1) (4 -6). VEGF is a potent angiogenic and vascular permeability factor that plays critical roles in both physiological and pathological angiogenesis (7). Recently, the expression of VEGF in response to heregulin-induced activation of the HER2/neu receptor tyrosine kinase in breast cancer cells (8), IGF-1 stimulation of colon cancer cells (9), and insulin treatment of retinal pigment epithelial cells (10) was shown to be mediated by HIF-1 via the phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways. Thus, HIF-1 regulates both hypoxia-and growth factor-induced VEGF expression.
Nitric oxide (NO) is known to mediate many physiological and pathological functions including vascular dilatation, cytotoxicity mediated by activated macrophages, and cGMP formation following glutamate receptor activation in neurons (18).
NO has also been implicated in pathological conditions such as destruction of tumor cells by macrophages, rheumatoid arthritis, and focal brain ischemia. There are several reports demonstrating that exposure of cells to certain NO donors or gaseous NO modulates HIF-1 activity (19 -23). S-nitrosoglutathione (GSNO) or NOC18 induces HIF-1 activity under nonhypoxic conditions (22). In contrast, sodium nitroprusside (SNP) inhibits hypoxia-induced HIF-1 activation (19 -21). However, the molecular mechanisms that regulate HIF-1␣ expression and transactivation in response to NO donors are poorly defined. In this study, we found that NOC18 induces HIF-1 activity by increasing HIF-1␣ protein synthesis via PI3K-and MAPK-dependent pathways.
Immunoblot Assays-Whole cell lysates were prepared by incubating cells for 30 min in cold radioimmune precipitation assay buffer containing 2 mM dithiothreitol, 1 mM NaVO3, and Complete protease inhibitor HEK293 cells were treated with NOC18 or DFX for 24 h, and total RNA was isolated. Expression of VEGF, GLUT1, and HIF-1␣ mRNA and 18 S rRNA was analyzed by RT-PCR.
Inhibitor Treatments-PD98059, LY294002, genistein, or rapamycin was added 1 h before exposure to NOC18 or 1% O 2 . CHX was added to the medium of HEK293 cells that were treated with NOC18, GSNO, or DFX for 4 h, and whole cell extracts were prepared at 15, 30, and 60 min.
RT-PCR-The protocol of RT-PCR is described elsewhere (33). Briefly, cells were lysed, and RNA was isolated with TRIzol reagent (Invitrogen). 0.5 g of total RNA were subjected to first strand cDNA synthesis using the SuperScript II RT kit (Invitrogen) with random hexamers. cDNAs were amplified with TaqGold polymerase in a thermal cycler with the following primer pairs: HIF1A, GAAAGCG-CAAGTCCTCAAA and CTATATGGTGATGATGTGGCACTA; VEGF, CCATGAACTTTCTGCTGTCTT and ATCGCATCAGGGGCACACAG; GLUT1, GGGCATGTGCTTCCAGTATGT and ACGAGGAGCACCGT-GAAGAT; 18 S, ATCCTGCCAGTAGCATATGC and ACCCGGGTTG-GTTTTGATCTG. For each primer pair, PCR was optimized for cycle number to obtain linearity between the amount of input RT product and output PCR product. Thermocycling conditions were 30 s at 94°C, 60 s at 57°C, and 30 s at 72°C for 25 (HIF1A), 27 (VEGF), or 14 (18 S rRNA) cycles preceded by 10 min at 94°C. PCR products were fractionated by 3% Nusieve agarose gel electrophoresis, stained with ethidium bromide, and visualized with UV.
Metabolic Labeling Assay-The protocol is described elsewhere (8). Briefly, a total of HEK293 cells were plated in a 10-cm dish, and 24 h later the cells were serum-starved for 20 h. The cells were pretreated with 500 M NOC18 or 100 M DFX for 30 min in methionine-free Dulbecco's modified Eagle's medium. [ 35 S]Met-Cys was added to a final concentration of 0.3 mCi/ml, and the cells were pulse-labeled for 20 -40 min and then harvested. Whole cell extracts were prepared, and 1 mg of extract was precleared with 60 l of protein A-Sepharose for 1 h. 20 l of anti-HIF-1 antibody H1 67 was added to the supernatant and rotated overnight at 4°C. 40 l of protein A-Sepharose was added, rotated for 2 h at 4°C, pelleted, and washed five times with 1 ml of radioimmune precipitation buffer. The samples were analyzed by SDS-polyacrylamide gel electrophoresis. The gel was dried and exposed to x-ray film.
Immunoprecipitation Assay-Cells were harvested in 200 l of lysis buffer (Dulbecco's PBS (pH 7.4), 0.1% Tween 20, 1 mM sodium orthovanadate, and Complete protease inhibitor) and drawn through a 20-gauge needle four times. The lysate was incubated on ice for 1 h, followed by centrifugation at 14,000 rpm for 15 min. The cleared lysates were brought to a volume of 1 ml with lysis buffer followed by a 2-h incubation with 20 l of anti-HA (Roche Applied Science) or anti-FLAG (Sigma) affinity matrix beads at 4°C on a rotator. The beads were then . Cells were exposed to 20 or 1% O 2 with or without NO donors for 16 h and then harvested for luciferase assays. C, cells were co-transfected with p2.1, pTK-RL, and the indicated amount of expression vectors encoding either no protein (EV) or a dominant negative form of HIF-1␣ (DN). The total amount of expression vectors was adjusted to 500 ng with empty vector. The ratio of firefly to Renilla luciferase activity (RLA) was determined and normalized to the value obtained from nonhypoxic cells transfected with empty vector to obtain the relative luciferase activity. Results shown represent mean Ϯ S.D. of three independent transfections.
washed three times with lysis buffer. Protein was eluted by the addition of Laemmli sample buffer and analyzed by SDS-PAGE and immunoblot analysis (16).
In Vitro HIF-1␣-VHL Interaction Assay-Glutathione S-transferase (GST)-HIF-1␣(429 -608) fusion protein was expressed in E. coli as described (16,33). Biotinylated methionine-labeled proteins were generated in reticulocyte lysates using the TNT T7 coupled transcription/ translation system using Transcend biotinylated tRNA (Promega). 25-g aliquots of HEK293 cell lysate were preincubated with NO donor or DFX for 30 min at 30°C, 2.5 g of GST-HIF-1␣(429 -608) was added, and the mixture was incubated for 30 min at 30°C. A 5-l aliquot of in vitro translated biotinylated VHL protein was mixed with 4 g of GST fusion protein in a final volume of 200 l of binding buffer (Dulbecco's PBS (pH 7.4), 0.1% Tween 20) and incubated for 2 h at 4°C with rotation followed by the addition of 10 l of glutathione-Sepharose 4B beads (Amersham Biosciences) and incubation at 4°C for 1 h. The beads were pelleted, washed 3 times in binding buffer, pelleted again, resuspended in Laemmli sample buffer, and analyzed by SDS-PAGE. Proteins were transferred to polyvinylidene difluoride membrane and visualized using streptavidin-labeled horseradish peroxidase and ECL reagent (Amersham Biosciences).
In Vitro Ubiquitination Assay-HEK293 cells were washed twice with cold hypotonic extraction buffer (20 mM Tris (pH 7.5), 5 mM KCl, 1.5 mM MgCl 2 , 1 mM dithiothreitol) and lysed in a Dounce homogenizer. The cell extract was centrifuged at 10,000 ϫ g for 10 min at 4°C, and the supernatant was stored at 70°C. Ubiquitination assays were performed as described previously (33). 2 l of HA-HIF-1␣ that was in vitro translated (TNT Quick Coupled Transcription/Translation System; Promega) in the presence of [ 35 S]methionine was incubated at 30°C in a volume of 40 l containing 27 l (50 g) of cell extract, 4 l of 10ϫ ATP-regenerating system (20 mM Tris (pH 7.5), 10 mM ATP, 10 mM magnesium acetate, 300 mM creatine phosphate, 0.5 mg/ml creatine phosphokinase), 4 l of 5 mg/ml ubiquitin (Sigma), 1 l of 150 M ubiquitin aldehyde (Sigma), and HA-HIF-1␣ was recovered using anti-HA-agarose beads, which were then mixed with SDS sample buffer and boiled for 5 min. The eluates were analyzed by SDS-PAGE and autoradiography.
Reporter Gene Assays-Reporter assays were performed in Hep3B cells and HEK293 cells (32,34). 5 ϫ 10 4 cells were plated per well on the day before transfection. In each transfection, the indicated dose of test plasmids, 200 ng of reporter gene plasmid, and 50 ng of the control plasmid pTK-RL (Promega), containing a thymidine kinase promoter upstream of Renilla reniformis (sea pansy) luciferase coding sequences, were premixed with Fugene 6 transfection reagent (Roche Applied Science). In each assay, the total amount of DNA was held constant by the addition of empty vector. After treatment, the cells were harvested, and the luciferase activity was determined using the Dual-Luciferase Reporter Assay System (Promega). The ratio of firefly to sea pansy luciferase activity was determined. For each experiment, at least two independent transfections were performed in triplicate.

NO Donors Activate HIF-1 under Nonhypoxic Conditions-
To study the effect of NO on HIF-1 activation, we tested several NO donors. NORs and NOCs spontaneously release NO with different kinetics (see "Experimental Procedures"), whereas GSNO and SNP require cellular thiol for NO release. HEK293 cells were exposed to the compounds for 1-4 h at 20% O 2 , harvested, and subjected to immunoblot analysis using anti-HIF-1␣ or anti-HIF-1␤ antibody (Fig. 1A). Neither NOR4 nor NOR5 induced HIF-1␣ protein accumulation (lanes 2-5). In contrast, exposure of cells to NOC18 or GSNO efficiently induced HIF-1␣ protein accumulation comparably with 100 M DFX (lanes 6 -8, 12, and 13). SNP did not induce HIF-1␣ accumulation (lanes 10 and 11). Expression of HIF-1␤ was not affected by NO donors or DFX. NOCs induced accumulation of HIF-1␣ with quite different kinetics as compared with GSNO. NOC18-induced accumulation was detected as early as 30 min and lasted no less than 8 h. The effect of GSNO peaked at 1 h (lane 8) and was lost by 4 h after the addition (lane 9).
NOC18 induced HIF-1␣ accumulation in a dose-dependent manner up to 500 M (Fig. 1B). Induction by GSNO was saturated at a concentration of 100 M. The accumulation of HIF-1␣ induced by NOC12, which releases NO by the same mechanism as NOC18 but has a different NO-releasing time constant, was stronger than that induced by NOC18 at 30 min at a concentration of 250 M (Fig. 1C, top panel). However, the induction of HIF-1␣ was dose-dependent such that the effect of 40 M NOC12 was weaker than 500 M NOC18 (Fig. 1C, bottom  panel).
We screened other cell lines for the effect of NO donors on HIF-1␣ protein levels. 500 M NOC18 induced HIF-1␣ accumulation in Hep3B human hepatocellular carcinoma cells and HCT116 human colorectal carcinoma cells as strongly as 100 M DFX (data not shown). NOC18 also induced HIF-1␣ in human umbilical vein endothelial cells (data not shown). These results indicate that the effect of NOC18 on HIF-1␣ expression is observed in multiple transformed and primary cell types.
We investigated by RT-PCR whether NO donors induced gene expression downstream of HIF-1. VEGF and GLUT1 mRNA expression was induced by NOC18 treatment under nonhypoxic conditions (Fig. 2). In contrast, HIF-1␣ mRNA expression was not affected by NOC18 treatment, indicating that the effect of NOC18 occurs at the level of HIF-1␣ protein expression. HEK293 cells were transfected with the reporter p2.1, containing a HIF-1-dependent HRE, or p2.4, containing a mutated HRE. NOC18 induced HRE-dependent gene expression in a dose-dependent manner comparably with DFX or 1% O 2 (Fig. 3, A and B). The mutated reporter p2.4 was not activated by NOC18 (Fig. 3B), and expression of a dominant negative form of HIF-1␣ reduced p2.1 reporter gene expression (Fig. 3C), providing evidence that the gene activation was HRE-and HIF-1-dependent. NOC18 also induced dose-dependent transcription of a reporter gene containing the VEGF promoter encompassing nucleotides Ϫ2274 to ϩ379 relative to the transcription start site (Fig. 3D).
NOC18 Does Not Prolong HIF-1␣ Protein Half-life-To determine whether NOC18 treatment affected HIF-1␣ protein half-life, HEK293 cells were treated with NOC18 or DFX for 4 h to induce HIF-1␣ expression, and then CHX was added to block ongoing protein synthesis. In the presence of CHX, the half-life of HIF-1␣ was Ͼ30 min in DFX-treated cells but Ͻ15 min in NOC18-treated cells (Fig. 4A). Similarly, the half-life of GSNO-induced HIF-1␣ is Ͻ15 min in the presence of CHX (Fig.  4B). These results indicate that HIF-1␣ expression in NOC18treated cells is dependent upon ongoing protein synthesis. Similar results were observed in Hep3B cells and human umbilical vein endothelial cells (data not shown).
To analyze the rate of HIF-1␣ synthesis, serum-starved HEK293 cells were pretreated with NOC18 or DFX for 30 min and then pulse-labeled with [ 35 S]Met-Cys for 20 or 40 min, followed by immunoprecipitation of HIF-1␣ (Fig. 4C). In contrast to control serum-starved cells (Fig. 4C, lane 1), 35 S-labeled HIF-1␣ was clearly increased in NOC18-treated cells (lanes 2 and 3), whereas the amount of labeled HIF-1␣ protein was not increased in cells treated with DFX (lanes 4). Thus, both the cycloheximide addition and metabolic labeling experiments provide evidence for increased synthesis of HIF-1␣ in response to NOC18 treatment. We also assayed the stability of a fusion protein, consisting of a nuclear localization signal (NLS), ␤-galactosidase sequences (encoded by the lacZ gene), and HIF-1␣ residues 548 -603. The NLS-LacZ-HIF1␣(548 -603) expression vector was transfected into HEK293 cells, and ␤-galactosidase activity was analyzed by X-gal staining after incubation of the cells in the presence of 500 M NOC18 or 100 M DFX. There was essentially no X-gal staining in cells that were transfected with empty vector, transfected with NLS-LacZ-HIF1␣(548 -603) without treatment, or transfected with NLS-LacZ-HIF-1␣(548 -603) with NOC18 treatment (Fig. 4D). In contrast, significant X-gal staining was detected in NLS-LacZ-HIF-1␣(548 -603)-transfected cells that were treated with DFX, which inhibits O 2 -dependent degradation mediated by the HIF-1␣ domain of the fusion protein.
NOC18 Does Not Affect the Interaction between HIF-1␣ and VHL in Vitro or in Vivo-Under hypoxic conditions, VHL-dependent ubiquitination of HIF-1␣ is inhibited (12)(13)(14). To determine whether NOC18 treatment affects ubiquitination, an in vitro assay was performed using lysates prepared from control and NOC18-treated cells. As shown in Fig. 5A, there was no significant difference detected between lysates from NOC18treated or untreated cells with respect to their ability to ubiquitinate HIF-1␣.
HEK293 cells were co-transfected with expression vectors encoding HA-tagged VHL and FLAG-tagged HIF-1␣. Aliquots of whole cell lysates were analyzed for expression of the proteins directly or following immunoprecipitation of HA-VHL or FLAG-HIF-1␣. HIF-1␣ was present in anti-HA immunoprecipitates from cells co-expressing HA-VHL and FLAG-HIF-1␣ (Fig.  5C, lane 3). Exposure of cells to NOC18 did not alter the interaction of HA-VHL and FLAG-HIF-1␣ (Fig. 5C, lanes 4 and  5), consistent with the inability of NOC18 to inhibit VHL and HIF-1␣ interaction in vitro. In contrast, DFX treatment inhibited the interaction (lane 6). FIH-1 is the asparagine hydroxylase that negatively regulates HIF-1␣ transactivation domain function under nonhypoxic conditions (16,17). The interaction between HA-FIH-1 and FLAG-HIF-1␣ was not affected by either NOC18 or DFX (Fig. 5D). Taken together, results presented in Fig. 5 indicate that the molecular mechanism of NOC18 action is distinct from the inhibition of hydroxylase activity that occurs in cells exposed to hypoxia or DFX.
Impact of NO Scavenger, Guanyl Cyclase Inhibitor, and Antioxidant on NOC18-induced HIF-1␣ Accumulation-To examine signal transduction pathways mediating effects of NO donors on HIF-1␣ protein induction, the NO scavenger carboxyl-PTIO was utilized (35). Carboxyl-PTIO significantly suppressed HIF-1␣ accumulation induced by NOC18 but not by DFX (Fig. 6A). Carboxyl-PTIO by itself did not have any effects. Next we examined the impact of guanylyl cyclase activity on HIF-1␣ accumulation. NO stimulates the activity of guanylyl cyclase, which catalyzes the production of cGMP, an important second messenger for signal transduction. In HEK293 cells, the specific guanyl cyclase inhibitor ODQ did not affect NOC18induced HIF-1␣ accumulation (Fig. 6B), providing evidence that the guanylyl cyclase-cGMP pathway does not contribute to HIF-1␣ accumulation induced by NOC18.
NO is a radical, and equimolar amounts of O 2 Ϫ and NO form peroxynitrite (ONOO Ϫ ), which decomposes at physiological pH to generate oxidant with similar reactivity to the hydroxyl radical. To examine whether the intracellular redox state modulates NOC18-induced HIF-1␣ accumulation, HEK293 cells were treated with NOC18 in the presence of 50 mM N-acetyl cysteine (NAC) (Fig. 6C). N-acetyl cysteine treatment did not affect HIF-1␣ levels, suggesting that thiol-mediated redox status does not play a critical role in NOC18-induced HIF-1␣ expression. Transient overexpression of the intracellular redox regulator thioredoxin also did not affect induction of HIF-1␣ expression by NOC18 (data not shown).
LY294002 and rapamycin inhibited expression of the HIF-1dependent reporter gene p2.1 induced by NOC18 but not by DFX, whereas genistein and PD98059 inhibited both NOC18and DFX-induced reporter gene expression (Fig. 8C, top). Interestingly, the stimulation of HIF-1␣ transactivation domain function by NOC18 was also blocked by kinase inhibitors, whereas only genistein blocked DFX-induced transactivation ( Fig. 8C, bottom). These results provide further evidence that NOC18 and DFX induce HIF-1 by distinct molecular mechanisms. Moreover, NOC18-induced HRE-dependent gene expression was suppressed by a dominant negative form of PI3K p85 subunit, AKT, or Ras, indicating critical roles of these signaling proteins in transducing the effects of NOC18 to HIF-1 (Fig. 8D).
NOC18-induced Activation of MAPK, PI3K, and Translational Regulators-HIF-1 activity induced by the stimulation of receptor tyrosine kinases or G protein-coupled receptors requires MAPK and PI3K signaling (10,36). To determine whether the MAPK and PI3K pathways were activated in NOC-18-treated cells, the phosphorylation of p42 ERK2 /p44 ERK1 and AKT were analyzed in HEK293 cells and HCT116 cells. Increased phosphorylation of p42 ERK2 /p44 ERK1 (Fig. 9A) and AKT (Fig. 9B) was induced by NOC18 treatment in both cell types.
The signal transduction pathway involving PI3K, AKT, and mTOR has been shown to regulate protein translation via phosphorylation of p70 S6K , the S6 ribosomal protein, and 4E-BP1. In both HCT116 cells (Fig. 10) and HEK293 cells (data not shown), the phosphorylation of p70 S6K , S6, and 4E-BP1 was induced by NOC18 stimulation in a dose-and time-dependent manner. The mRNA cap-binding protein eIF-4E was also phosphorylated by NOC18 treatment of HCT116 cells (Fig. 10). This result is consistent with studies indicating that ERK activates the MAPK signal-integrating kinases, MNK1 and MNK2, which in turn phosphorylate eIF-4E (37,38). DISCUSSION The studies reported above demonstrate that treatment of several different cell types with the NO donor NOC18 induces HIF-1␣ protein expression and HIF-1 transcriptional activation, resulting in VEGF and GLUT1 mRNA expression. NOC18 treatment did not increase the half-life of HIF-1␣ protein, did not inhibit the interaction between HIF-1␣ and VHL, and did not inhibit the ubiquitination of HIF-1␣, indicating that the mechanism of NOC18 action does not involve inhibition of HIF-1␣ prolyl hydroxylation. Rather than increasing the stability of HIF-1␣, the data suggest that NOC18 increases the rate of HIF-1␣ protein synthesis.
Whereas exposure of cells to hypoxia or DFX decreases HIF-1␣ protein degradation, exposure of cells to heregulin, IGF-1, insulin, or prostaglandin E 2 increases HIF-1␣ protein synthesis (8 -10, 36). In previous studies of MCF-7 and HCT116 cells, the effect on protein synthesis was documented by cycloheximide inhibition and by pulse-chase experiments (8). In the present study, we also confirmed that NOC18 treatment stimulated the synthesis of HIF-1␣ but had no effect on HIF-1␣ protein stability in HEK293 cells. Thus, as in the case of growth factor-treated cells, the increased expression of HIF-1␣ protein in NOC18-treated cells is due to increased synthesis.
As previously observed in growth factor-treated cells, the effect of NOC18 is dependent upon its activation of the PI3K and MAPK pathways. Dependence on MEK activity for phosphorylation of 4E-BP1 and p70 S6K has been demonstrated in other cellular contexts. In the case of IGF-1-stimulated colon cancer cells, both MEK and PI3K are required for activation of p70 S6K , with MEK inhibitors preventing the phosphorylation of Thr-421/Ser-424 in the Thr-389 by mTOR (39). ERK has been shown to phosphorylate 4E-BP1 in vitro (40). The MEK-ERK pathway also stimulates the phosphorylation of eIF-4E, which is required for its mRNA cap binding activity (37). Thus, NOC signaling both derepresses (via phosphorylation of 4E-BP1) and activates (via phosphorylation of eIF-4E and p70 S6K ) protein synthesis. The effects of NO donors may not be specific for HIF-1␣. The known targets for phosphorylation by mTOR are regulators of protein synthesis. The translation of several FIG. 9. MAPK and PI3K pathway signaling in NOC18-treated cells. HEK293 and HCT116 cells were exposed to 100 or 500 M NOC18. Whole cell lysates were prepared after 15, 30, or 60 min and subjected to immunoblot (IB) assays using antibodies specific for phosphorylated (Thr-202/Tyr-204) or total p42 ERK2 /p44 ERK1 MAPK (A) and phosphorylated (Ser-473) or total AKT (B).
FIG. 10. Phosphorylation of the translational regulators p70 S6K , S6 ribosomal protein, and eIF-4E in NOC18-treated HCT116 cells. Cells were serum-starved for 24 h prior to NOC18 treatment. Whole cell extracts were prepared after NOC18 stimulation and subjected to immunoblot (IB) assays using antibodies specific for phosphorylated (Thr-421/Ser-424) or total p70 S6K , phosphorylated (Ser-235/236) or total S6 ribosomal protein (S6R), phosphorylated (Ser-65) or total 4E-BP, and phosphorylated (Ser-209) or total eIF-4E. dozen different mRNAs are known to be regulated by this pathway, and sequences in the 5Ј-untranslated region of the respective mRNAs may determine the degree to which the translation of any particular mRNA can be modulated by mTOR signaling. HIF-1␣ protein expression is likely to be particularly sensitive to changes in the rate of synthesis because of its extremely short half-life under nonhypoxic conditions.
HIF-1 activity is regulated not only by HIF-1␣ protein expression but also by HIF-1␣ transcriptional activity. Our data analyzing transactivation mediated by Gal4-HIF-1␣-TAD fusion proteins demonstrate that NOC18 treatment also induces HIF-1␣ TAD activity under nonhypoxic conditions. A regulatory switch controlling TAD activity involves O 2 -dependent hydroxylation of Asn-803 by FIH-1. NOC18 treatment did not promote dissociation of FIH-1 and HIF-1␣. TAD activity is also regulated by a MAPK-dependent mechanism (41). The MEK-1 inhibitor PD98059 blocked NOC18-induced HIF-1 activation and NOC18-induced MAPK activation, suggesting a link between NOC18, MEK/ERK, and HIF-1␣. Published data suggest that the direct target of MEK/ERK may be the coactivators CREB-binding protein and p300, which interact with the TADs (42).
The action of NO in biological systems can be mediated directly by NO or by conversion of NO to NO Ϫ or NO ϩ equivalents (43). Because two enzymes in the ubiquitin-proteasome pathway, E1 and E2, contain thiols in their active sites, these thiols were a priori candidates as targets of NO donors. However, our experimental results do not support this mechanism of action for NOC18. Another potential target is HIF-1␣ itself, since there is a report that GSNO induces nitrosylation of HIF-1␣ (44). However, our results indicate that if NOC18 induces nitrosylation of HIF-1␣, this modification does not lead to accumulation of the protein.
NOC18 treatment had no effect on the interaction of HIF-1␣ and VHL, whereas GSNO partially inhibited the interaction, and SNP dramatically augmented the interaction. SNP may stimulate the prolyl hydroxylation-ubiquitination system and promote increased HIF-1␣ degradation. Consistent with this hypothesis, SNP inhibited HIF-1␣ accumulation induced by DFX. Thus, different NO donors activate or inhibit HIF-1 through different molecular mechanisms.
Recent studies have demonstrated that NO donors stimulate cellular signaling cascades (45)(46)(47). Overexpression of a dominant negative form of Ras significantly inhibited NOC18-induced HRE-dependent gene expression, and the tyrosine kinase inhibitor genistein almost completely abolished NOC18induced HIF-1␣ expression, suggesting that one or more protein-tyrosine kinases or phosphatases may be regulated by nitrosative modification. NOC18 treatment also induced phosphorylation of both AKT and ERK. Thus, NOC18 treatment modulates protein kinase signaling pathways similar to the effects of growth factor treatment. Determination of the extent to which NO signaling to HIF-1 participates in physiological and pathophysiological processes will require further investigation.