Correspondence to Robert S. Freeman: Robert_Freeman{at}urmc.rochester.edu
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Introduction |
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A number of reports have shown that reducing O2 to as low as 1% can increase cell survival in vitro (Yun et al., 1997). Nonetheless, cells maintained in culture are typically exposed to O2 levels that far exceed those measured in vivo. For example, whereas normal tissue O2 levels range from 1 to 5% in the adult mammalian brain (Erecinska and Silver, 2001), dissociated neurons are routinely maintained in an atmosphere of 5% CO2 and 95% air, equivalent to 20% O2. Although reducing O2 tension can impact a wide range of biochemical processes (Bickler and Donohoe, 2002), a particularly well-characterized response recently implicated in neuronal cell survival involves activation of the transcription factor hypoxia-inducible factor (HIF; Halterman et al., 1999; Zaman et al., 1999; Piret et al., 2002; Soucek et al., 2003).
There are three isoforms of HIF, each consisting of a common ß subunit and a unique subunit; of these, HIF-1
is the best characterized (Semenza, 1999). When O2 is not limiting, HIF-1
readily associates with the von Hippel Lindau protein, part of an E3 ubiquitin ligase, and is rapidly polyubiquitinated and degraded by the proteasome. The interaction between HIF-1
and von Hippel Lindau protein is mediated by the O2-dependent hydroxylation of two proline residues in HIF-1
(Ivan et al., 2001; Jaakkola et al., 2001; Yu et al., 2001). The enzymes that catalyze this reaction are members of the egg-laying nine (EGLN) family of prolyl hydroxylases (Bruick and McKnight, 2001; Epstein et al., 2001). In hypoxic cells, EGLN prolyl hydroxylase activity is reduced leading to stabilization of HIF-1
and transactivation of a large and diverse group of HIF-responsive genes (for review see Safran and Kaelin, 2003).
Here, we show that exposing sympathetic neurons to low O2 during NGF deprivation significantly reduces their rate of cell death. Induction of BIMEL and loss of mitochondrial cytochrome c were both suppressed in NGF-deprived neurons exposed to low O2. Forced expression of BIMEL restored cytochrome c release but did not reverse the protective effect of low O2, suggesting that additional mechanisms were important for inhibiting cell death. Results from several experiments implicated HIF as a potential mediator of the neuroprotective effect of low O2. This was confirmed by microinjection experiments combining targeted deletion of HIF-1 with ectopic expression of BIMEL in the same neurons. These results provide a new model for how O2 tension influences the apoptotic events that underlie trophic factor deprivationinduced death.
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Results |
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Induction of BIMEL after NGF withdrawal is suppressed by low O2
Because the BCL-2 family proteins BAX and BIMEL are critical for cytochrome c release after NGF withdrawal, we compared their protein levels before and after NGF withdrawal in neurons cultured under standard and reduced O2. We also analyzed expression of the pro-survival protein BCL-XL and for changes in the phosphorylation of c-Jun. Phosphorylation of c-Jun increases in neurons deprived of NGF under standard culture conditions (Ham et al., 1995; Virdee et al., 1997) and we observed an essentially identical increase in neurons exposed to low O2 (Fig. 3 A). BAX and BCL-XL protein levels remained unchanged after 20 h of NGF withdrawal, regardless of O2 tension. BIMEL protein levels, on the other hand, increased an average of fourfold when neurons were deprived of NGF under standard O2 conditions; but when NGF deprivation was performed at 1% O2, a much smaller increase in BIMEL was observed (Fig. 3, A and B; also see Fig. 4 B). This effect appeared to be specific because low O2 did not inhibit c-Jun phosphorylation or decrease the expression of any of the other proteins examined.
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Similar to BIMEL protein, the increase in BIMEL mRNA during NGF deprivation was also suppressed by low O2 (Fig. 4 C). Both c-Jun and the FOXO family member FKHRL1 have been implicated in up-regulating BIMEL transcription in cells deprived of NGF (Putcha et al., 2001; Harris and Johnson, 2001; Whitfield et al., 2001; Gilley et al., 2003). c-Jun is activated relatively soon after NGF withdrawal in part through JNK-dependent phosphorylation of Ser-63 (Virdee et al., 1997; Eilers et al., 1998). Activation of FKHRL1 after NGF withdrawal involves the dephosphorylation of sites originally phosphorylated by Akt protein kinase (Zheng et al., 2002; Gilley et al., 2003). Thus, FKHRL1 activation typically coincides with inactivation of Akt (Burgering and Kops, 2002).
To test whether the JNK/c-Jun and Akt/FKHRL1 pathways might be altered in neurons kept under different O2 conditions, we immunoblotted c-Jun and Akt using antibodies specific for their phosphorylated and activated forms. In neurons maintained at 20% O2, the amount of phosphorylated c-Jun increased within 5 h of NGF removal and remained elevated for at least 20 h. Conversely, phosphorylated Akt levels decreased within 5 h of NGF deprivation and remained low (unpublished data). These same changes in c-Jun and Akt phosphorylation occurred when the experiment was performed on neurons exposed to 1% O2 (Fig. 4 D), suggesting that the reduced O2 tension used in these studies is unlikely to impact either c-Jun activation or Akt inactivation after NGF withdrawal.
We also examined whether low O2 might directly influence FKHRL1 by altering its phosphorylation at Thr-32, a site targeted by Akt (Brunet et al., 1999). As shown previously (Gilley et al., 2003), neurons deprived of NGF under standard O2 conditions had reduced levels of Thr-32phosphorylated FKHRL1 compared with neurons maintained with NGF (Fig. 4 E). Unexpectedly, the amount of FKHRL1 phosphorylated at this site did not decrease when NGF was withdrawn from neurons exposed to 1% O2, despite the concurrent decrease in Akt phosphorylation. These results suggest that activation of FKHRL1 in response to NGF withdrawal might be inhibited in neurons exposed to reduced O2. Such a scenario, if corroborated, could help explain the suppression of BIMEL mRNA expression that occurs under these conditions (Gilley et al., 2003).
Ectopic BIMEL reverses the ability of low O2 to block cytochrome c release but not cell death
The results described above suggest a model in which low O2 inhibits cell death by suppressing BIMEL induction, which in turn inhibits the release of cytochrome c from mitochondria and ultimately cell death. As a test of this idea, we infected neurons with a control adenovirus expressing EGFP or one expressing a BIMEL/EGFP fusion protein. The next day, the cells were deprived of NGF and switched to a 1% O2 environment for an additional 24 h, after which the cells were examined by immunofluorescence for cytochrome c localization. Parallel cultures were deprived of NGF for 48 h to assess the effects of BIMEL expression on cell survival. The majority of control EGFP-expressing neurons exposed to low O2 during NGF deprivation retained intense, punctate cytochrome c immunofluorescence (Fig. 5, A and B), similar to the results shown with uninfected neurons in Fig. 2. In contrast, very few of the NGF-deprived neurons exposed to low O2 and expressing BIMEL/EGFP retained punctate cytochrome c labeling. Despite their apparent lack of mitochondrial-localized cytochrome c, the majority of cells expressing BIMEL/EGFP remained healthy 48 h after withdrawing NGF (Fig. 5 C). Thus, whereas forced expression of BIMEL can overcome the block to cytochrome c release, there must be additional processes induced by low O2 (apparently downstream or independent of cytochrome c release) that can sustain cell survival in the absence of NGF.
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To determine whether HIF-1PP
AG can influence trophic factor deprivationinduced cell death, neurons were infected with Ad-HIF-1
PP
AG/EGFP or Ad-EGFP and the next day deprived of NGF. By 24 h of NGF deprivation, about half of the Ad-EGFP infected and uninfected neurons showed signs of chromatin condensation and nuclear fragmentation (Fig. 7, A and B). In contrast, the nuclei of nearly all HIF-1
PP
AG/EGFP expressing neurons appeared healthy, closely resembling those of NGF-maintained neurons. By 48 h, over 80% of neurons expressing HIF-1
PP
AG/EGFP remained healthy compared with just 25% of the control cells. Survival was also assessed using a MTT metabolic activity assay. NGF-deprived neurons infected with Ad-HIF-1
PP
AG/EGFP retained significantly more MTT reducing activity than control neurons infected with Ad-EGFP (Fig. 7 C). Thus, expression of a stabilized, prolyl hydroxylase-resistant form of HIF-1
protects neurons, at least transiently, from apoptosis caused by trophic factor deprivation.
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Low O2-mediated protection is partially reversed by disrupting HIF-1 expression and completely abrogated when disruption of HIF-1
is combined with ectopic BIMEL
The activation of HIF after exposure to low O2 and the ability of mutant HIF-1PP
AG to inhibit death after NGF withdrawal led us to test the significance of HIF-1
for the neuroprotective effect of low O2. For these experiments, we isolated sympathetic neurons from transgenic mice homozygous for a loxP flanked (floxed) HIF-1
gene (HIF-1
fl/fl; Ryan et al., 2000). Previous studies have shown that introducing Cre recombinase into HIF-1
fl/fl cells results in efficient deletion of the HIF-1
gene and loss of HIF-1-dependent gene induction (Ryan et al., 2000; Seagroves et al., 2001).
To determine whether expression of Cre recombinase in HIF-1fl/fl sympathetic neurons results in a substantial loss of HIF-1
, we infected the neurons with an adenovirus that expresses Cre (Ad-Cre) or with the control Ad-EGFP virus. Immunofluorescence confirmed Cre expression in virtually all of the cells by 24 h after infection (unpublished data). RT-PCR analysis revealed greatly reduced HIF-1
expression specifically in Ad-Cre infected cultures (Fig. 8 A), suggesting that efficient deletion of the floxed HIF-1
gene had occurred. Loss of HIF-1
had no effect on the ability of NGF to promote survival for at least 4 d, and initial NGF deprivation experiments suggested that HIF-1
deletion did not significantly affect short-term protection afforded by low O2 as measured at 24 h after NGF withdrawal (unpublished data; see below).
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Discussion |
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Certain events triggered by NGF withdrawal were not affected by lowering O2. For example, dephosphorylation of ERKs and Akt and activation of JNKs (as judged by c-Jun phosphorylation) occurred similarly in neurons exposed to 20% or 1% O2. These findings differ from those obtained in models of ischemic and hypoxic preconditioning in which neuroprotection requires activation of Akt (Ruscher et al., 2002; Wick et al., 2002). In our paradigm, lowering the O2 concentration interrupted the apoptotic program subsequent to JNK activation, resulting in reduced BIMEL expression. A similar situation has been reported in colon cancer cells, where O2 deprivation was found to down-regulate expression of the pro-apoptotic BCL-2 family protein BID (Erler et al., 2004).
Although the nature of the downstream block to cell death remains uncertain, several observations point to a role for HIF. First, HIF was activated in NGF-deprived neurons exposed to low O2. Second, introducing HIF-1PP
AG mimicked the downstream block by inhibiting caspase-3 activation and cell death without preventing cytochrome c release. Third, whereas the neuroprotective effect of low O2 was partially reduced in neurons lacking HIF-1
, protection was completely lost in neurons expressing ectopic BIMEL (to override the first block) and lacking HIF-1
.
How might low O2 inhibit caspase activation after cytochrome c release? One possibility is that neurons exposed to low oxygen may express or maintain higher levels of proteins capable of blocking caspase activation. For example, Potts et al. (2003) reported that down-regulation of the X-linked inhibitor of apoptosis protein (XIAP) was necessary for caspase activation and cell death after NGF withdrawal, even after cytochrome c was present in the cytoplasm. Thus, an increase in XIAP expression under reduced O2 conditions might be sufficient to inhibit trophic factor deprivationinduced death. A similar mechanism was recently suggested based on the induction of IAP-2 expression in oxygen-deprived kidney cells (Dong et al., 2003). Additional mechanisms for blocking cell death after cytochrome c release may involve down-regulation of Apaf-1 or downstream caspases (Jia et al., 2001; Devarajan et al., 2002). We are currently investigating whether the expression level of any of these proteins is influenced by lowering O2 tension. Although our efforts so far have focused on well-studied pro-survival and pro-apoptotic pathways, it is important to realize that reducing O2 tension will at some point influence numerous other biochemical processes. Cellular effects of low oxygen can include changes in ion channel function, reactive O2 species generation, and energy metabolism, as well as alterations in the activity of a large number of enzymes (Bickler and Donohoe, 2002).
In cells maintained under standard culture conditions of 20% O2, HIF activity is kept at an extremely low level do to the highly efficient degradation of its subunits. This occurs in large part because the O2-dependent enzymes responsible for targeting HIF-
subunits for ubiquitination (HIF prolyl hydroxylases) have a Km for O2 that is near its concentration in air (Hirsilä et al., 2003). Because HIF prolyl hydroxylase activity is reduced in cells at physiologic O2 tensions (Epstein et al., 2001), basal HIF activity in most cells in vivo should be substantially greater than in cells in culture. Given this scenario together with a neuroprotective role for HIF-1, down-regulating basal HIF activity may be an important part of the overall cell death program. How could inactivation of HIF-1 be ensured in neurons deprived of NGF? In a previous study, we showed that NGF deprivation results in an increase in the expression of the SM-20 gene (Lipscomb et al., 1999). Recently, SM-20 was found to be the rat orthologue of EGLN3, one of three HIF prolyl hydroxylases in mammals (Freeman et al., 2003). Thus, up-regulation of SM-20/EGLN3 could serve to quench an HIF-dependent neuroprotective pathway. At O2 tensions that are too low to support EGLN3 activity, this mechanism might be compromised resulting in sustained HIF activation and inhibition of cell death (Freeman et al., 2004).
In summary, basic cellular mechanisms controlling cell death and survival are generally characterized in cells maintained under supraphysiologic oxygen conditions. As shown here, some of these mechanisms can be influenced by reducing O2 tension. We believe these results highlight the importance of carefully defining the O2-sensitivity of mechanisms that determine cell viability.
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Materials and methods |
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Cell culture
Primary cultures of sympathetic neurons were prepared from the superior cervical ganglia of embryonic day-21 Sprague-Dawley rats (Harlan) or newborn HIF-1fl/fl mice (Ryan et al., 2000) as described elsewhere (Lipscomb et al., 1999). After preplating to enrich for neurons, cells were plated on collagen- or polyornithine/laminin-coated dishes in NGF-containing media (90% MEM, 10% FBS, 2 mM L-glutamine, 20 µM uridine, 20 µM fluorodeoxyuridine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 50 ng/ml NGF). For NGF deprivation, neurons were rinsed twice with PBS before addition of media lacking NGF and containing neutralizing anti-NGF antiserum. All treatments were initiated 56 d after plating. The caspase inhibitor BAF (50 µM) was included in the media for immunoblotting and cytochrome c immunofluorescence experiments. BAF treatment prevents terminal phases of cell death without blocking upstream events including JNK activation, BIMEL up-regulation, and cytochrome c release (Deshmukh and Johnson, 1998; Tsui-Pierchala et al., 2000; Putcha et al., 2001). O2 tension was controlled by incubating cells at 37°C in humidified, O2/CO2-regulated incubators (Thermo Forma) adjusted to 5% CO2 and the indicated O2 tension (balanced by N2), or in a standard CO2-controlled incubator maintained at 5% CO2/95% air (equivalent to
20% O2). In these experiments, a relative O2 tension of 1% (in the gas phase) was equivalent to a partial pressure of O2 of
6.97.3 mm Hg. Note that the cells were continuously exposed (uninterrupted) to reduced oxygen for times that greatly exceeded the 12 h needed to equilibrate the culture medium with the O2 in the gas phase.
Assessing viability and caspase activity
After removal from the incubator, cells were quickly rinsed with PBS and then fixed in 4% PFA. The cultures were then stained with Hoechst 33,342 (1 µg/ml in PBS) and visualized under phase-contrast and epi-fluorescence using a 40x objective and a microscope (model Diaphot 300; Nikon). Viability was assessed based on the appearance of the neurons and the Hoechst-stained nuclei (Lipscomb et al., 2001). Healthy neurons had round, smooth, and refractile cell bodies with clearly discernible nuclear membranes and nucleoli, and diffuse Hoechst-stained chromatin. In contrast, dying or dead cells were characterized by condensed or undetectable chromatin and fragmented and atrophied cell bodies. For experiments with recombinant adenoviruses, 150200 neurons expressing EGFP or HIF-1PP
AG/EGFP were scored for each treatment per experiment. MTT assays (Maggirwar et al., 1998) and caspase-3 activity assays (Straub et al., 2003) were described previously.
Immunofluorescence
Immediately after removal from the incubator, cells were fixed in 4% PFA for 30 min at 4°C. Immunofluorescence was done as described previously (Lipscomb et al., 2001) using a 1:500 dilution of mouse anticytochrome c antibody (BD Biosciences) or a 1:1000 dilution of anti-Cre antibody followed by a TRITC-conjugated goat antimouse secondary antibody (Jackson ImmunoResearch Laboratories) diluted 1:300. Cells were rinsed with PBS before staining with Hoechst 33,342. In all NGF-maintained neurons, the cytochrome c immunofluorescence pattern is punctate throughout the cytoplasm and in neuronal processes but excludes the nucleus. After 20 h of NGF deprivation done in the presence of BAF, all of the neurons exhibited a faint fluorescence signal that was evenly distributed throughout the cytoplasm and nucleus. None of the cells showed an obvious partial loss of punctate staining. For each experiment, the pattern of cytochrome c immunofluorescence was scored by an observer blinded to the experimental treatments. Digital images were captured using a microscope (model Diaphot 300; Nikon) equipped with a DAGE-MTI CCD camera and Scion Image software (Scion Corp.).
Immunoblotting
Cells were rinsed once with ice-cold PBS and solubilized in SDS-PAGE sample buffer immediately after removal from the incubator. Lysates were boiled for 10 min and centrifuged for 5 min at 12,000 g before separation by SDS-PAGE. Separated proteins were transferred to nitrocellulose membranes, which were blocked in TBS containing 5% nonfat dry milk and 0.1% Tween-20 (TBST) for 1 h at RT. Membranes were incubated overnight at 4°C in the same buffer containing the following primary antibodies at the indicated dilutions: anticytochrome c, 1:500 (BD Biosciences); anti-BIM, 1:1,000 (StressGen Biotechnologies); anti-BAX, 1:1,000 (Upstate Biotechnology); antiBCL-XL, 1:1,000 (Santa Cruz Biotechnology); antiphospho-c-Jun (Ser-63), 1:1,000 (Cell Signaling); antiphospho-Akt (Ser-473), 1:1,000 (Cell Signaling); anti-Akt, 1:1,000 (Cell Signaling); antiphospho-ERK1/2, 1:1,000 (Cell Signaling); anti-ERK1/2, 1:1,000 (Cell Signaling); antiphospho-FKHRL1 (Thr-32), 1:1,000 (Upstate Biotechnology); anti-actin, 1:300 (Sigma-Aldrich); antiHIF-1, 1:1,000 (Novus Biologicals). The blots were then incubated for 1 h with appropriate HRP-conjugated secondary antibodies diluted 1:10,000 in TBST and detected by ECL using Super Signal West Dura substrate (Pierce Chemical Co.). To detect endogenous HIF-1
in sympathetic neurons, cells were lysed in RIPA buffer [50 mM Tris, pH 8.0, 150 mM NaCl, 1% NP-40, 1% deoxycholate, 0.1% SDS, 1 mM PMSF, 5 µg/ml leupeptin, and 5 µg/ml aprotinin] and the lysates immunoprecipitated as described previously (Lipscomb et al., 2001) using 2 µg of antiHIF-1
antibody. Immunoprecipitated proteins were then immunoblotted using the same antiHIF-1
antibody. For all immunoblotting experiments, the data shown are representative of the results from three or more independent experiments.
RTPCR
Total RNA was extracted from equal numbers of neurons plated on collagen-coated 6-well dishes using an RNeasy Mini kit (QIAGEN). Semi-quantitative RT-PCR to analyze BIMEL and cyclophilin expression was performed as described previously (Estus et al., 1994). PCR products shown in Fig. 4 C were labeled by incorporation of -[32P]-dCTP over 21 and 17 cycles of amplification, respectively. HIF-1
and cyclophilin PCR products in Fig. 8 A were amplified over 30 and 25 cycles, respectively, and analyzed on agarose gels stained with ethidium bromide.
Plasmids and site-directed mutagenesis
The plasmid containing HRE-luciferase was a gift from G. Semenza (Johns Hopkins University, Baltimore, MD) and R. Ratan (Harvard Medical School, Boston, MA). For expression of HIF-1 and the HIF-1
/EGFP fusion protein, the ORF of human HIF-1
was generated by PCR using Pfu Turbo DNA polymerase (Stratagene) and confirmed by DNA sequencing. The HIF-1
cDNA was subcloned into pcDNA3 and pEGFP-C1 (CLONTECH Laboratories, Inc.). Site-directed mutagenesis of HIF-1
and HIF-1
/EGFP was done using QuickChange (Stratagene) to change Pro-402 and Pro-564 to Ala and Gly. The resulting plasmids, HIF-1
PP
AG and HIF-1
PP
AG/EGFP, were verified by DNA sequencing.
Ectopic gene expression
Replication-deficient adenovirus vectors were produced using the AdEasy system (Qbiogene) following the manufacturer's protocol. EGFP and HIF-1PP
AG/EGFP were subcloned into pShuttle-CMV; the HRE-luciferase cassette was subcloned into pShuttle. Recombination between the pShuttle-derived plasmids and pAdEasy-1 was performed in BJ5183 E. coli (Qbiogene). Recombinant plasmids were confirmed by restriction digests, amplified in DH5
E. coli, and then linearized with PacI before transfection into HEK293 cells. Recombinant replication-defective adenoviruses were plaque purified and verified by PCR and immunoblotting using antibodies against HIF-1
or EGFP. High titer viral stocks (
1010 plaque-forming units/ml) were obtained after amplification in HEK293 cells and purification through CsCl gradients. The purified virus stocks were diluted to the indicated multiplicity of infection (MOI) in complete medium and then added to cells using half the normal culture volume. The medium was replaced after 12 h. Microinjections were performed as described previously (Lipscomb et al., 2001). Plasmid DNAs (50100 µg/ml) were diluted in injection buffer containing 100 mM KCl, 10 mM potassium phosphate, pH 7.4, and 4 mg/ml rhodamine-dextran (to identify injected cells).
Luciferase assays
Luciferase activities were determined using the Dual-Luciferase Reporter Assay system (Promega) and a TD 20/20 luminometer (Turner) following the manufacturer's protocol.
Online supplemental material
COS-7 cells were maintained in 90% DME and 10% FBS. The pcDNA3-based plasmid expressing rat EGLN3 (also called SM-20) was described previously (Lipscomb et al., 2001). Transfections were done using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. Adenovirus infections, immunoblotting, and luciferase assays were performed as described above. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200407079/DC1.
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Acknowledgments |
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This work was supported by National Institutes of Health grants NS34400 and NS42224.
Submitted: 13 July 2004
Accepted: 20 December 2004
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References |
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