From the Institute of General Pathology, Catholic
University Medical School, 00168 Rome, Italy, ¶ Department of
Neuroscience, The Johns Hopkins University School of Medicine,
Baltimore, Maryland 21205,
Medical Research Council (MRC)-Dunn
Human Nutrition Unit Wellcome Trust/MRC Building Hills Road,
Cambridge CB2 2XY, United Kingdom, and ** Department of
Chemistry, University of Otago, P. O. Box 56, Dunedin,
New Zealand
Received for publication, January 31, 2003
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ABSTRACT |
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Reactive oxygen species (ROS) act as
both signaling molecules and mediators of cell damage in the nervous
system and are implicated in the pathogenesis of neurodegenerative
diseases. Neurotrophic factors such as the nerve-derived growth factor
(NGF) support neuronal survival during development and promote
regeneration after neuronal injury through the activation of
intracellular signals whose molecular effectors and downstream targets
are still largely unknown. Here we present evidence that early
oxidative signals initiated by NGF in PC12 cells, an NGF-responsive
cell line, play a critical role in preventing apoptosis induced by serum deprivation. This redox-signaling cascade involves
phosphatidylinositol 3-kinase, the small GTPase Rac-1, and the
transcription factor cAMP-responsive element-binding protein (CREB), a
molecule essential to promote NGF-dependent survival. We
found that ROS are necessary for NGF-dependent
phosphorylation of CREB, an event directly correlated with CREB
activity, whereas hydrogen peroxide induces a robust CREB
phosphorylation. Cells exposed to NGF show a late decrease in the
intracellular content of ROS when compared with untreated cells and
increased expression of the mitochondrial antioxidant enzyme manganese
superoxide dismutase, a general inhibitor of cell death. Accordingly,
serum deprivation-induced apoptosis was selectively inhibited by low
concentrations of the mitochondrially targeted antioxidant Mito Q
(mitoquinol/mitoquinone). Taken together, these data demonstrate that
the oxidant-dependent activation of CREB is a component of
NGF survival signaling in PC12 cells and outline an intriguing
circuitry by which a cytosolic redox cascade promotes cell survival at
least in part by increasing mitochondrial resistance to oxidative stress.
Reactive oxygen species
(ROS)1 such as hydrogen
peroxide (H2O), superoxide anion (O In apparent contradiction with the role of ROS in human and
experimental pathology, a large body of evidence indicates that oxygen
species, when produced in low amounts and in a controlled fashion, are
not toxic and can function as physiological components of the signaling
generated by cytokine and growth factors (7, 8). ROS, and hydrogen
peroxide in particular, act as critical signaling molecules in cellular
signaling generated by a wide array of stimuli, including tumor
necrosis factor- Although the molecular mechanisms of oxygen radical-mediated signaling
is still largely unclear, much evidence suggests that ROS strongly
regulates protein tyrosine phosphorylation by directly inhibiting
protein-tyrosine phosphatase activity (13, 14). Moreover, the small
GTPase Rac-1 (15), whose activity is linked to ROS generation by the
combined effect on membrane oxidases and metabolism of arachidonic acid
(16), mediates cellular responses to oxidative stimuli. In neurons
oxidant species such as NO (17) and ROS regulate signaling events,
including PI 3-kinase/Akt pathway, c-Jun NH2-terminal
kinase/stress-activated protein kinase pathway, and activation of the
nuclear factor Target-derived neurotrophins, including nerve growth factor (NGF) and
brain-derived neurotrophic factor act as major regulators of neuronal
cell differentiation and survival in the developing nervous system and
exert a protective effect after acute neuronal injury (20, 21).
Neurotrophins exert their effects through a complex network of
intracellular signals involving the activation of the PI 3-kinase/Akt
pathway, Ras/mitogen-activated protein kinase pathway, and the small
GTPases of the Rho family (Rho, Rac, and CDC42) (20, 22). Many
NGF-dependent survival signals converge on the
transcription factor cAMP-responsive element-binding protein (CREB), a
key molecule responsible for the expression of antiapoptotic genes in
sympathetic neurons and cerebellar granule cells (23, 24).
Only few target genes for neurotrophin signaling have been identified
so far; because neurotrophins increase cell resistance to oxidative
stress (25), and application of NGF to sympathetic neurons reduce the
generation of oxygen radicals in mitochondria upon serum withdrawal
(26), it is conceivable that these trophic factors modulate cellular
antioxidant defense. Transcriptional regulation of the mitochondrial
superoxide scavenger Mn-SOD represents a general protective event in
response to cellular stress and to pro-apoptotic stimuli (27-30).
Interestingly, a cAMP-responsive element is present within the Mn-SOD
promoter, and 12-O-tetradecanoylphorbol-13-acetate-mediated gene expression in lung carcinoma cell line (28) is dependent upon CREB activation.
Here we show that induction of Mn-SOD in serum-deprived PC12 cells is
NGF- and CREB-dependent and may therefore represent a
general modality of neurotrophin signaling. Importantly, the molecular
pathway linking NGF to Mn-SOD expression reveals an important role for
ROS in triggering CREB activation and gene expression, thereby
outlining a redox circuitry in which cytosolic oxidants are used as
messenger intermediates to increase mitochondrial protection from
oxidative stress.
Plasmids, Antibodies, and Chemicals--
Myristylated human Akt
expression construct was kindly provided by Dr. A. Bellacosa.
Expression constructs for CREB-VP16 and CREB-VP16/
The following antibodies were used in the present study:
anti-phospho-Akt Ser-473, anti-phospho-Akt Thr-308, anti-Akt,
anti-phospho-FKHR Ser-256, and anti-phospho-extracellular
signal-regulated kinase 1/2 (New England Biolabs), anti-extracellular
signal-regulated kinase, anti-phospho-CREB Ser-133, anti-Mn-SOD
(Upstate Biotechnology), anti-actin (Santa Cruz Biotechnology).
Horseradish peroxidase (HRP)-conjugated reagents were from Amersham
Biosciences (anti-mouse IgG/HRP), Bio-Rad (anti-rabbit IgG/HRP), and
Chemicon (anti-goat IgG/HRP).
Rat nerve growth factor was a kind gift of Drs. D. D. Ginty and
D. Mercanti. Dichlorofluorescein diacetate (DCF-DA) and
dihydroethidium (DHE) were from Molecular Probes, 2-methoxyestradiol
(2-ME), hydrogen peroxide, N-acetylcysteine, and propidium
iodide were purchased from Sigma. The synthetic mitochondrial
antioxidant Mito Q, described in Kelso et al. (43), was
obtained from Drs. M. Murphy and R. Smith. Transfection reagent
LipofectAMINE was purchased from Promega.
Cell Lines--
PC12 were maintained in RPMI 1640 with 10%
fetal bovine serum and 10% horse serum in collagen-coated cell culture
dishes. 293T kidney adenocarcinoma cells, and the derivative
ecotropic packaging line Phoenix (kindly provided by Dr. S. Lowe) were maintained in Dulbecco's modified Eagle's medium plus 10%
fetal bovine serum. The E1A/Ras-transformed, SOD2 +/+ and Cell Transfection and Infection--
PC12 cells and E1A/Ras MEFs
were transfected with LipofectAMINE (Promega) according to the
manufacturer's instructions. Average transfection efficiency was
15-20%, based on cell positivity for GFP. Cells were left to recover
for 24-48 h after transfection before functional or biochemical
assays. 293T and Phoenix cells were transfected by calcium-DNA
co-precipitation according to the standard procedure (50% efficiency).
For retroviral infection, PC12 cells were grown for 48 h in the
supernatant of Phoenix cells previously transfected with retroviral (pLPC, pLPC-RacN17, pLPC-Mn-SOD/AS) constructs. Transduced cells were
enriched by 48-h selection in puromycin (2.5 µg/ml).
For in vitro transformation, SOD +/+ and SOD2 Measurement of ROS--
For detection of reactive oxygen
species, cells were maintained for 16 h in low (0.5%) serum.
After medium replacement with Hanks' balanced salt solution or
serum-free RPMI, NGF was added, and cells were incubated for an
additional 60 or 120 min. During the last 30 min of incubation, DCF-DA
(1 µg/ml) or DHE (10 µM) was added to cells. After
quick detachment from the plate, cells were immediately subjected to
flow cytometry (FL-1 for DCF-DA and FL-2 for DHE) using a COULTER-EPICS
flow cytometer equipped with an argon laser lamp (emission 488 nm). The
mean cell fluorescence values were expressed as raw data or as
fluorescence variations (stimulated
In some experiments, cell were plated in serum-free medium with or
without NGF, and the fluorescent probes were added directly to the
medium 24 h later for 30 min. Cells were then processed as
described above.
Assays for Cell Viability--
Cells were seeded in serum-free
medium with or without NGF and/or antioxidants. In experiments with
Mito Q, cell were pretreated with the drug for 8 h in complete
medium before serum withdrawal. 48 h later, cell viability was
determined by flow cytometry.
Live and dead cells were distinguished by flow cytometry according to
two criteria, (a) forward/side scatter profile and
(b) exclusion of propidium iodide. The majority of cells
positive for propidium iodide (dead cells) fell in a distinct region on the forward/side scatter histogram (R2), which could also be easily identified in the forward/side scatter plot in the absence of the
fluorescent marker. This latter population was found to be slightly more abundant than the population of propidium iodide-positive cells, likely due to the presence of early apoptotic cells non permeable to propidium iodide. Cell debris was gated out in all the
measurements. The percentage of surviving cells was determined according to the forward/scatter profile, with the following formula: [% cells in the "live" region R1/(% cells in "dead" region
R2 + % cells in the "live" region R1)] × 100.
In some experiments GFP was expressed in cells together with a gene of
interest to allow the identification of transfected cells by flow
cytometry. Because GFP can leak out of apoptotic cells, the absolute
number of live, GFP-positive cells was then determined using
fluorescent microspheres (Flow-Count fluorospheres, Coulter) as an
internal standard, according to the manufacturer's recommendations.
Cell Stimulation and Lysis--
PC12 cells were stimulated in 0 or 0.5% fetal bovine serum with NGF and in 0% FCS with hydrogen
peroxide for the indicated times after 16 h of incubation in low
(0.5%) serum. Antioxidants, when necessary, were added either 16 h (N-acetylcysteine (NAC)) or 5 h (Mito Q) before stimulation.
For Western blot analysis cells were lysed in lysis buffer (150 mM NaCl, 50 mM Tris-HCl, pH 8, 2 mM
EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml
aprotinin, leupeptin, and pepstatin, 1 mM sodium orthovanadate) containing 1% Triton X-100 and 0.1% SDS; lysates were
cleared by centrifugation, and equal amounts of protein for each sample
were subjected to SDS-PAGE followed by electrotransfer, immunoblotting,
and ECL detection. Band quantitation was performed using the Quantity.
One software (Bio-Rad). For total RNA extraction, cells were lysed in
Trizol (Invitrogen) and further processed according to the
manufacturer's recommendations.
For "in gel" determination of SOD activities, cell pellets were
resuspended in phosphate buffer, pH 8, containing 1 mM
phenylmethylsulfonyl fluoride and sonicated. Equal amounts of protein
were loaded on a non-denaturing polyacrylamide gel and processed as
described (46).
Northern Blot Analysis of Mn-SOD mRNA--
15-20 µg of
total RNA were subjected to Northern blot analysis. The rat Mn-SOD
cDNA and the rat actin cDNA were obtained by reverse
transcription-PCR and random-prime-labeled using the Redi-Prime labeling kit (Amersham Biosciences). After hybridization for Mn-SOD (65 °C overnight) filters were stripped and reprobed for actin to
verify that comparable amounts of RNA had been loaded in all lanes.
NGF Rescues PC12 Cells from Apoptosis Induced by Serum
Withdrawal--
PC12 cells are dependent on calf serum for survival
in vitro and rapidly undergo apoptosis when placed in
serum-free medium (25). The addition of NGF to the culture medium
largely prevents death induced by serum starvation, with 70% of the
cells remaining viable after 48 h of fetal bovine serum
withdrawal (Fig. 1, A and B).
NGF activates a multiplicity of signaling cascades in PC12 cells,
mainly through the interaction with the tyrosine kinase receptor TrkA
and the low affinity receptor p75 (20). As previously shown, NGF
addition to serum-deprived PC12 cells dramatically induces the
phosphorylation of Akt/protein kinase B and extracellular signal-regulated kinases 1/2 (Fig. 1C), an event mirroring
the increase in enzymatic catalytic activity. Although phosphorylation of mitogen-activated protein kinases likely reflects the activation of
the Ras/Raf pathway by NGF, Akt is a direct target for the lipid
products of the PI 3-kinase, a major mediator of survival signaling by
NGF (31). Overexpression of a membrane-targeted, constitutively active
mutant of human Akt nearly completely abolishes cell death induced by
serum deprivation (Fig. 1D). Interestingly, a negligible or
at best marginal effect was upon transfection of an active form of Raf
(CAAX-Raf, data not shown), further suggesting that
NGF-mediated cell survival in serum-deprived condition is mainly
mediated by the PI 3-kinase/Akt pathway (24).
NGF Modulates the Intracellular Content of ROS in PC12
Cells--
Reactive oxygen species generated in mitochondria trigger
apoptotic cell death in response to many stimuli (32). Moreover, ROS
are involved in regulation of gene expression, proliferation, and cell
differentiation induced by cytokine and growth factor receptors
(7).
NGF treatment of PC12 cells maintained in the absence of serum is
initially (15 min to 2 h) accompanied by increased intracellular content of ROS (Fig. 2, A and
B), as evaluated by the oxidation of two different
redox-sensitive fluorescent probes, DCF-DA (Fig. 2, A and
B, a) and DHE (Fig. 2B, b).
Because DCF-DA is mainly sensitive to hydrogen peroxide, whereas DHE
detects superoxide (33), it is likely that both reactive species are
rapidly produced in response to NGF. During the first 2 h of NGF
treatment, the increase of intracellular ROS is
concentration-dependent, significant at 10 ng/ml and more
pronounced at 100 ng/ml (Fig. 2A). Pretreatment of PC12
cells with the glutathione precursor and radical scavenger NAC
drastically reduced early NGF-dependent oxidative events
(Fig. 2A, b), and a similar decrease was also
observed in cells transfected with a dominant negative mutant of GTPase
Rac-1 (N17Rac1) (Fig. 2A, c).
Interestingly, the early increase of ROS after NGF treatment is
followed by a decrease of intracellular oxidants (both peroxides and
superoxide) with respect to untreated cells at 24 h after NGF
application (Fig. 2B, a and b). The
late antioxidant effect of NGF coincides with the onset of
morphological signs of death in untreated serum-starved cells. This
observation suggests that elevated levels of oxidants detected 24 h after serum deprivation in PC12 cells are causally linked to cell
death and are counteracted by application of NGF.
NGF Signaling to Akt and CREB Requires ROS--
Upon treatment
with NGF, PC12 cells respond with a robust activation of the PI
3-kinase pathway, which couples receptor engagement to cell survival
through the activation of Akt/protein kinase B (34, 36). A critical
downstream event in the survival signaling initiated by NGF is
represented by the phosphorylation of CREB, a transcription factor
necessary and sufficient for neurotrophin-dependent survival (23, 35, 36). Accordingly, CREB may be a direct target of Akt
(24, 37).
To determine whether the early burst of ROS plays a role in
NGF-dependent signaling to Akt and CREB, PC12 cells were
treated with glutathione precursor and the ROS scavenger NAC to prevent intracellular oxidations, and cell signaling ability was assessed. Removal of ROS resulted in a dramatic decrease of Akt phosphorylation and activity, as evaluated by Ser-256 phosphorylation of the Akt substrate FKHR (38) (Fig. 3A).
Concomitantly, pretreatment of PC12 cells with antioxidant NAC resulted
in a decrease of CREB phosphorylation on its critical residue, Ser-133,
as revealed by immunoblotting using a phosphorylation site-specific
antibody (Fig. 3A). Moreover, inhibition of Rac activity, an
event necessary for NGF-dependent redox signaling (Fig.
2A, c), resulted in inhibition of CREB
phosphorylation (Fig. 3B). Because CREB phosphorylation is
critical for transcriptional activation (39), our data suggest that
CREB activity in response to NGF could also be impaired.
To further test the possibility that redox signals regulates CREB in
PC12 cells, we assessed whether exogenous hydrogen peroxide is
sufficient to induce CREB activation. Cells exposed to 1 mM H2O2 show increased activation of CREB and Akt,
to an extent comparable with NGF (Fig. 3C). Hydrogen
peroxide-dependent Akt and CREB phosphorylation is
inhibited by the PI 3-kinase inhibitor wortmannin (Fig. 3D), suggesting that oxidants activate multiple survival pathways upstream of PI 3-kinase.
NGF Induces Mn-SOD Expression in PC12
Cells--
CREB-dependent transcriptional activation of
antiapoptotic genes contributes to the survival effect induced by NGF.
Late reduction of intracellular ROS in NGF-treated PC12 cells (Fig.
2B, a and b) suggests that
neurotrophins might regulate antioxidant enzyme expression.
Interestingly, the mitochondrial protein Bcl2, which may have some
antioxidant functions, is among the few NGF-dependent antiapoptotic proteins hitherto identified and is modulated in a
CREB-dependent fashion.
The mitochondrial antioxidant enzyme and putative survival factor
Mn-SOD is induced in many cell types by cytokines (tumor necrosis
factor-
Exposure of serum-deprived PC12 cells to NGF results in a significant
increase of expression of Mn-SOD mRNA transcripts of 4.1 and 1.1 kilobases (Fig. 4A). SOD2
mRNA induction is present 1 h after NGF treatment and peaks at
12 and 24 h after induction. An increase in RNA transcripts is
mirrored by the increase of both Mn-SOD protein (Fig. 4B)
and enzyme activity, as assessed by an in-gel nitro blue
tetrazolium-based SOD assay (Fig. 4C) (42). Accordingly, the
intracellular concentration of superoxide is significantly decreased
24 h after NGF stimulation (Fig. 4D). Taken together,
these data suggest that Mn-SOD is a downstream target of NGF signaling
in PC12 cells.
Modulation of Mn-SOD Expression by PI 3-Kinase and CREB--
PI
3-kinase and the transcription factor CREB play a pivotal role in
mediating antiapoptotic signaling generated by NGF. PC12 cell
pretreatment with the PI 3-kinase inhibitor wortmannin significantly inhibited NGF-induced Mn-SOD expression (Fig.
5A). Similarly, pretreatment
of PC12 cells with NAC, which diminished both ROS production and CREB
phosphorylation in response to NGF (see Figs. 2 and 3), resulted
in a significant decrease of Mn-SOD transcription (Fig. 5A).
These data suggest that NGF-dependent induction of Mn-SOD
requires both PI 3-kinase activity and oxidants in a fashion that
mirrors CREB phosphorylation.
To determine whether CREB is sufficient to induce Mn-SOD mRNAs, we
overexpressed a constitutively active form of CREB (VP-16 CREB) (23) in
both PC12 (Fig. 5B, a) and 293T cells (Fig.
5B, b). Activation of CREB resulted in a
significant increase of Mn-SOD mRNAs when compared with cells
overexpressing a mutant form of CREB lacking the DNA binding domain
( Redox Regulation of NGF-dependent Cell
Survival--
Our data suggest that NGF regulates ROS in a complex
fashion such that oxygen species generated early after neurotrophin
stimulation signal to CREB and act as pro-survival mediators. Later on,
when toxic oxidants are generated, NGF contributes to their elimination through up-regulation of compartment-specific, mitochondrial
antioxidant, such as Mn-SOD. To assess the physiological relevance of
these redox changes in PC12 cells, we used two different antioxidants, the generic, membrane-permeant scavenger NAC and a
mitochondria-targeted derivative of ubiquinol (Mito Q) (43). PC12 cells
treated with 10 mM NAC, a condition that severely impairs
NGF signaling to Akt and CREB (Fig. 3A), showed decreased
ability to survive in both the presence and the absence of NGF (Fig. 6,
A and B). It is therefore conceivable that NAC
promotes cell death by blocking protective redox signals initiated by
NGF. Conversely, cell exposure to Mito Q mimicked NGF treatment and
resulted in an increased resistance to cell death (Fig.
6A, gray columns).
Because Mito Q is present at a low concentration (1 µM)
in the culture medium and preferentially accumulates in mitochondria
(43), it is unlikely to interfere with oxidative reactions that take
place in the cytosol, where early ROS are thought to be produced.
Still, Mito Q selectively removes harmful oxidants leaking from the
respiratory chain during apoptosis, thereby promoting cell survival. In
line with this view, Mito Q, unlike NAC, had no effect on
NGF-dependent CREB phosphorylation (Fig. 6C) nor
did it block the early rise of ROS in cells treated with NGF (data not
shown). Along similar lines of evidence, we found that cell exposure to
the recently described SOD inhibitor 2-ME (33) drastically reduced
NGF-dependent survival without significantly affecting
cells grown in standard medium (10% fetal bovine serum) (Fig.
6D). Taken together, these data suggest that oxidative
events generated during cell survival and cell death are distinct and
physically compartmentalized between cytosol and mitochondria and that
NGF survival signaling is critically dependent on the elimination of
intracellular superoxide.
Mn-SOD Is Required for Trk-mediated Antiapoptotic
Signaling--
To confirm that antioxidant defense against
mitochondrial oxidants is important for antiapoptotic signaling by NGF,
we first inhibited expression of Mn-SOD by retrovirus-mediated
expression of an antisense cDNA (pBabe/Mn-SODAS), as assessed by
Western blot analysis (Fig.
7A, b). PC12 cells
expressing low levels of Mn-SOD (Mn-SODAS) were more sensitive to cell
death in the presence of NGF when compared with mock-transfected cells
(Fig. 7A, a). The important role of Mn-SOD in NGF
trophic signaling was further demonstrated by making SOD2-deficient
transformed fibroblasts or their wild-type counterparts responsive to
the neurotrophin through transfection of the NGF receptor Trk.
Transformed fibroblasts are highly sensitive to apoptosis by serum
deprivation (44). As indicated in Fig. 7B, NGF signaling
through Trk protects SOD2-proficient cells from serum withdrawal,
whereas no significant protection was observed SOD2 null cells. A block
of trophic signaling in SOD2 Experimental observations described in the present paper lead to
two major conclusions as follows. 1) Reactive oxygen species have a
role in transducing survival signals released by NGF in PC12 cells.
Because PC12 cells respond to NGF at least in part through the Trk-A
receptor, the above finding is in line with a growing body of evidence
concerning the involvement of ROS in intracellular signaling downstream
of PTK-receptor molecules. 2) The up-regulation of mitochondrial
superoxide dismutase (Mn-SOD) is part of the antiapoptotic genetic
program triggered in PC12 cells by nerve growth factor. This is a novel
insight that expands our limited knowledge regarding the
transcriptional targets of NGF. Moreover, the above observation
indicates that mitochondrial protection from oxidative stress is a
potentially general mechanism of action for neurotrophins in
physiological and pathological settings.
We have postulated that ROS produced shortly after cell exposure to NGF
serve signaling functions downstream of activated NGF receptor(s). This
idea is in agreement with recent reports on redox signaling by NGF
receptors in neuronal cells (45, 46). Although not biochemically
characterized in detail, these oxidant species include probably both
hydrogen peroxide and superoxide, based on the data obtained with
fluorescent redox probes with different radical specificity (DCF-DA and
DHE). It should be noted that probe oxidation in response to NGF was
not prevented by the NO synthase inhibitors
L-N-monomethylarginine and diphenyleneiodonium (not shown), ruling out the possibility that DCF-DA and DHE are oxidized by NO. Interestingly, NGF-dependent generation of
ROS involves the small GTPase Rac-1, a general transducer for growth factor signaling repeatedly implicated in ligand-dependent
generation of oxygen species and in the promotion of cell survival
(47), already reported to mediate neurite outgrowth (48) and cell differentiation in cells of neuronal origin exposed to NGF. Although the present data suggest a role for Rac-induced ROS in survival signaling by NGF, it is conceivable that other
NGF-dependent signals involving Rac utilize, at least to
some extent, oxygen species as messenger intermediates. To this end, it
is noteworthy that oxygen radicals can also induce cell cytoskeleton
reorganization (49) and that differentiation of at least some types of
neuronal cells involves the generation of oxygen species (45).
An important point emerging from these studies is that NGF signaling to
AkT and CREB requires ROS and is severely impaired upon antioxidant
treatment. Although experiments with oxidized NAC (data not shown)
confirm that the inhibitory effect of N-acetylcysteine on
the NGF cascade is directly linked to the capacity of the compound to
lower the intracellular concentration of ROS, the link between oxygen
species and antiapoptotic signaling is further strengthened by the fact
that exogenous hydrogen peroxide, as already reported (50), is
sufficient per se to induce site-specific phosphorylation of
Akt and CREB.
The redox modulation of CREB phosphorylation/activation represents an
intriguing finding whose molecular mechanism deserves further
investigation. As a first hypothesis, it is conceivable that Akt, once
activated by oxidants, directly phosphorylates CREB, a possibility
supported by the observation that both Akt and CREB phosphorylation in
response to H2O2 are PI
3-kinase-dependent (Fig. 3C and Ref. 47).
Another important question is whether redox modulation of CREB operates
as well in the process of NGF-dependent neuronal
differentiation, because it has been reported that ROS induced by NGF
are required for differentiation of PC12 cells (45), a process clearly
dependent on CREB activity (51).
The finding that a redox-sensitive antiapoptotic cascade is triggered
in PC12 cells by NGF may also have broader pathophysiological implications. In fact, because the nervous system is highly prone to
oxidative damage (4), activation of survival pathways by oxidants may
represent a sensitive and efficient safeguard mechanism designed to
increase cell resistance to environmental stress in response to early
changes in the intracellular redox balance.
This hypothesis is supported by the finding here presented that NGF
induces expression of Mn-SOD, an enzyme able to remove harmful
oxidants, that such induction is dependent on PI 3-kinase activity, and
that interference with Mn-SOD expression results in impaired NGF
survival signaling in both PC12 cells and Trk-expressing transformed
fibroblasts (Fig. 7). Moreover, Mn-SOD, which is a well recognized
antiapoptotic enzyme promoting mitochondrial integrity, has already
been shown to protect other cell types from apoptosis by serum
deprivation (30, 33), and a decrease in mitochondrial superoxide has
been proposed as a mechanism for neuroprotection by NGF (26).
Furthermore, signs of neurodegeneration have been reported in the brain
of SOD2-deficient mice (6). Interestingly, another well established
target for NGF survival signaling, Bcl-2 (23), is also located in
mitochondria, which underlines the importance of these organelles as
targets for neurotrophin signaling and suggests a possible physical
and/or functional interaction between the two survival proteins.
Although the mechanism of Mn-SOD induction by NGF deserves further
investigation, our data imply CREB in the NGF-dependent regulation of Mn-SOD. In fact, an ideal CREB-responsive element is
present at position The proposed role for ROS as both mediators and targets for survival
signaling by NGF may appear contradictory. We suggest that oxygen
species involved in early NGF signaling are different from those
responsible for cell death in both their timing of production and their
subcellular sites of production. Although NGF-induced ROS are produced
rapidly after receptor stimulation through Rac-1-dependent
mechanisms, late generation of ROS likely takes place in mitochondria
and reflects the onset of mitochondrial dysfunction. These latter
oxidants may be eliminated by Mn-SOD, which is in turn induced by NGF
(Fig. 8). Importantly, although Mn-SOD activity is expected to lead to
peroxide accumulation, decreased DCF-DA fluorescence in concomitance
with elevated intracellular SOD is not surprising and is consistent
with important recent reports (54, 55).
Although in part speculative, the idea that NGF-triggered redox
signaling is intracellularly compartmentalized is in line with similar
findings concerning survival signaling by tumor necrosis factor-
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, interleukin-1 (9), platelet-derived growth factor,
epidermal growth factor, insulin (Ref. 7 and references therein),
vascular endothelial growth factor (10), antigens, mitogenic lectins
(11), and in growth control by cell-cell contact (12).
B (18, 19), suggesting a dual role as endogenous
toxins and signaling molecules.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
LZ were obtained
from Dr. D. D. Ginty. Rac-1 N17 cDNA was provided by Dr. A. Hall and subcloned in the EcoRI site of the pLPC retroviral
expression vector (a gift of Dr. S. Lowe). The retroviral construct
encoding the oncogenes V12 H-Ras and E1A on the pLPC backbone was
provided by Dr. Lowe. The rat Mn-SOD cDNA was obtained by reverse
transcription-PCR from PC12 total RNA and cloned in antisense
orientation in the EcoRI site of pLPC. Plasmid encoding the
green fluorescent protein (GFP) under the transcriptional control of
the cytomegalovirus promoter was purchased from Promega.
/
MEF
lines were derived by in vitro transformation of primary
embryonic fibroblasts from either wild-type or SOD2
/
C57Bl 6J mice
(kind gifts of Dr. C. J. Epstein, University of California,
San Francisco, CA). Transformed clones of each genotype were
transfected with the rat Trk cDNA (provided by Dr. Ginty), selected
in puromycin, and assessed for Trk expression by flow cytometry.
Wild-type and mutant cells expressing comparable levels of the receptor
were chosen for further experimental analysis.
/
MEFs
were infected with the pLPC-E1A/H-Ras construct, which harbors no
selectable marker, and left in culture for 2 weeks. Transformed foci
were then isolated and further expended.
unstimulated).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
PC12 cell death upon serum withdrawal and
rescue by NGF. A, forward/scatter-based analysis for
cell survival of PC12 cells. R1, live cells;
R2, dead cells. Most of R2 cells stained positive for
propidium iodide (PI, not shown). B, drastic
reduction of PC 12 cell survival after 48 h in serum-free medium
(white column versus light gray column) is
largely prevented by NGF (dark gray column). The percentage of live and
dead cells was determined by flow cytometry, as described under
"Experimental Procedures." % of surviving cells was calculated as
(% of cells in R1/(% of cells in R1 plus % of cells in R2)) × 100. The histogram is representative of several independent
experiments. C, phosphorylation of Akt (pAkt) and
extracellular signal-regulated kinases (pMAPK) induced by
NGF in serum-starved PC-12 cells. Equal protein loading was verified by
reversible Ponceau S staining of the nitrocellulose filters and by
immunostaining for total (phosphorylated + unphosphorylated)
extracellular signal-regulated kinase 1/2 and Akt. D,
membrane-targeted (myristoylated) AkT substitutes NGF for PC12 cell
rescue from serum withdrawal. Cell were co-transfected in complete
medium with either empty PcDNA3 vector (Neo) or
PcDNA3/MyrAkT in a 4:1 ratio with a vector encoding the green
fluorescent protein downstream of the cytomegalovirus promoter. 24 h after transfection equal numbers of cells were plated in serum-free
medium with or without NGF. 48 h later, absolute numbers of
GFP-positive, live cells were determined by flow cytometry using
Flow-Count fluorospheres of known concentration as internal standard.
Values are expressed as % of cell counts in the presence of FCS (100%
survival) and are the mean ± S.D. of duplicate samples. The
histogram is representative of two independent experiments.
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Fig. 2.
Modulation of intracellular ROS by NGF in
serum-deprived PC12 cells. A, a,
dose-dependent increase of intracellular peroxides in PC12
cells exposed to NGF for 60 min. DCF-DA was added 30 min before reading
at 20 µg/ml in Hanks' balanced salt solution. Probe oxidation was
quantified by flow cytometry, as described under "Experimental
Procedures." Values are mean fluorescence (mean ± S.D. of
duplicate samples) in arbitrary units (a.u.). The figure is
representative of several independent experiments. b,
N-acetylcysteine inhibits the early raise of intracellular
peroxides induced by NGF. PC12 cells were incubated for 16 h with
10 mM NAC in serum-free medium, stimulated with 100 ng/ml
NGF for 30 min, loaded with DCF-DA for an additional 30 min, and
subjected to flow cytometry. Values are fluorescence variations
(NGF-Control (Ctrl)) (mean ± S.D. of duplicate
samples). c, reduced generation of peroxide in response to
NGF in PC12 cells expressing an inhibitory mutant of Rac-1 (N17Rac).
Cells were retrovirally transduced with RacN17 or the corresponding
empty vector (pLPC), left to recover for 24 h, and selected in
puromycin for an additional 48 h. After overnight incubation in
0.5% FCS, cells were switched to serum-free medium, stimulated with
NGF for 30 min plus an additional 30 min in the presence of 20 µg/ml
DCF-DA, and subjected to flow cytometry as described above. Values are
variations (stimulated unstimulated) of mean cell
fluorescence and are the mean ± S.D. of duplicate samples. The
figure is representative of two independent experiments. It should be
noted that the percentage of transduced cells was never higher than
60%, based on GFP expression, even after the selection step in
puromycin. B, opposite effect of NGF of intracellular
oxidations at early and late time points. PC12 cells were incubated for
24 h in serum-free medium with or without NGF (NGF 24 h).
Alternatively, NGF was added to cells for only 2 h before flow
cytometry (NGF 2 h). Cells were loaded for 30 min before analysis
with DCF-DA (20 µg/ml) (a) or with dihydroethidium (10 µM) (b) directly in the serum-free medium.
Values are variations of cell mean fluorescence (NGF stimulated
unstimulated). Histograms are representative of two
independent experiments.
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Fig. 3.
Requirement of ROS for NGF signaling to AkT
and CREB. A, effect of NAC on NGF signaling to AkT and
CREB. Cells were preincubated as described in Fig. 2A,
b, with 10 mM NAC or left untreated. After
16 h, NGF was added for 15 min at the indicated concentrations.
Cells were quickly lysed, and protein extracts were subjected to
SDS-PAGE, electrotransfer, and immunoblotting with the indicated
phospho-specific antibodies. Equal protein loading throughout the gel
was verified by reversible Ponceau S staining or anti-actin
immunostaining. The bands of interest (Ser-473 + Thr-308 AkT, Ser-256
FKHR, Ser-133 CREB) are indicated by arrows. p-,
phosphorylated. B, inhibition of Rac-1 attenuates CREB
phosphorylation by NGF. Cell were processed as in Fig. 2A,
c, until stimulation with 100 ng/ml for the indicated times.
After cell lysis, protein extracts were subjected to SDS-PAGE and
immunoblotting with anti-phospho-Ser-133 CREB. Equal protein loading
was verified by reversible Ponceau S staining and by anti actin
immunoblotting of the same nitrocellulose filter. Densitometric band
intensities for Ser(P)-133 CREB are indicated. C,
phosphorylation of AkT and CREB in PC12 cells exposed to hydrogen
peroxide. Cells were incubated for 16 h in RPMI, 0.5% FCS,
switched to serum-free Hanks' balanced salt solution, and stimulated
with either 100 ng/ml NGF or 1 mM hydrogen peroxide for the
indicated times. Analysis of protein phosphorylation was performed as
described in B. Equal protein loading was verified by
anti-actin immunoblotting. D, effects of
H2O2 on AkT and CREB are inhibited by
wortmannin (WO). Serum-starved PC12 cells were incubated for
10 min with 10 µM wortmannin or the relative vehicle
(Me2SO); hydrogen Peroxide was then added for 15 min, and
protein phosphorylation was analyzed as described above. Wortmannin was
occasionally found to increase the base-line phosphorylation and
activity (as assessed by FKHR phosphorylation) of AkT, whereas
peroxide-induced phosphorylation was consistently and significantly
reduced.
, interleukin-1) (27, 29) and growth factors (platelet-derived growth factor, vascular endothelial growth factor) (40, 41) through signaling cascades that often involve ROS and
redox-sensitive transcriptional regulators (36, 52). Moreover, analysis
of the human Mn-SOD gene promoter region has revealed the presence of a
perfect CRE, a binding site for CREB, which accounts for gene response
to 12-O-tetradecanoylphorbol-13-acetate in a human lung
carcinoma cell line (28).
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Fig. 4.
Transcriptional up-regulation of Mn-SOD by
NGF in serum-depleted PC12 cells. A, time course for
Mn-SOD mRNA expression in PC12 cells exposed to NGF. Cells were
preincubated in low (0.5%) serum for 16 h before exposure to NGF
in serum-free medium for the indicated times. Total mRNA was
extracted and subjected to Northern blot analysis. Two waves of
induction at 1 and 12 h, respectively, were observed. The two main
transcripts of 4.1 and 2.1 kb are indicated by arrows. Equal
RNA loading was verified by filter re-hybridization for a housekeeping
gene (glyceraldehyde 3-phosphate dehydrogenase). Fold induction
(F.I.) with respect to the unstimulated sample was
determined by band densitometry after normalization for the
housekeeping gene. B and C, increases in Mn-SOD
immunoreactive protein (B) and in-gel activity
(C) in response to NGF. In B band intensities are
indicated. In C, a slight increase of Mn-SOD activity in
serum-starved cells in comparison to cells grown in standard medium was
observed, in agreement with previous reports (30, 32). Note that
CuZn-SOD, unlike the mitochondrial, manganese-dependent
enzyme, is marginally or not affected by serum deprivation and NGF.
Ctrl, control. D, a reduction in intracellular
superoxide accompanies up-regulation of Mn-SOD by NGF. Intracellular
superoxide was measured as in Fig. 2 24 h after serum withdrawal
and incubation with (NGF 24 h) or without (ctrl) NGF.
The figure is representative of several independent experiments.
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Fig. 5.
Effect of PI 3-kinase and CREB on Mn-SOD gene
induction. A, induction of Mn-SOD by NGF is blocked or
attenuated by the PI 3-kinase inhibitor wortmannin (Wo) and
by NAC. Cells were preincubated with wortmannin (10 µM)
for 10 min and with NAC for 16 h before exposure to NGF for the
indicated times. The two Mn-SOD transcripts are indicated by
arrows. The filter was stripped and re-hybridized for actin,
to ensure equal RNA loading. Ctrl, control. B,
active CREB is sufficient to induce Mn-SOD in PC12 and 293 T cells.
Cells were transfected by LipofectAMINE and calcium/DNA
co-precipitation, respectively, and incubated for 48 h in complete
medium. 293T cells were also exposed to NAC 10 mM for
24 h before RNA extraction (NAC+). 20 µg total RNA were
subjected to Northern blot analysis as in A. In
B, b, ribosomal RNA was used as the loading
control. Transfection efficiencies for the two cell lines are
indicated.
LZ-VP16, Fig. 5B, b). Taken together these
data suggests that CREB is required to regulate Mn-SOD expression.
Importantly, induction of Mn-SOD by active CREB is insensitive to NAC
(5B, b), suggesting that NAC does not indiscriminately repress Mn-SOD gene and reinforcing the idea that
signaling roles for ROS in the NGF cascade are upstream of CREB activation.
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Fig. 6.
Redox regulation of NGF-dependent
cell survival. A, modulation of serum-starved PC12 cell
survival by NAC and Mito Q. Shown are variations in the percentage of
cell survival in antioxidant- versus vehicle-treated cells
with (black columns) or without (gray columns)
NGF 100 ng/ml. NAC reduces cell survival mainly in NGF-treated cells,
whereas Mito Q increases cell resistance to serum deprivation, with
marginal effects on NGF. B, raw percentages of cell survival
in one of two independent experiments. A small inhibitory effect of
Me2SO (vehicle for Mito Q) was consistently observed, maybe
due to its properties of radical scavenger. NAC (10 mM) was
added to cells upon serum deprivation together with NGF, whereas Mito Q
was added 8 h earlier in complete medium to allow accumulation in
active mitochondria and removed for the rest of the experiment.
C, NAC, but not Mito Q, interferes with NGF signaling to
CREB. Cells were treated as in A, except that Mito Q was
added for 8 h in serum-free medium. Phosphorylation of CREB
(P-CREB) was assessed as described above. Equal protein
loading was verified by anti-actin immunoblotting of the same
nitrocellulose filter. D, inhibition of NGF rescue by the
SOD inhibitor 2-ME. Cells were seeded in either standard medium (10%
FCS) or serum-free medium plus 100 ng/ml NGF (SF + NGF), in
presence of titrated concentrations of 2-ME. Cell survival was
determined 48 h later by flow cytometry. Fig. representative of
two independent experiments. Values are mean ± S.D. of duplicate
samples.
/
cells occurred downstream of
NGF-induced CREB phosphorylation, which occurred at comparable levels
in both cell lines (Fig. 7B, b). Although in part
artificial, the MEF/Trk model strongly confirms the involvement of
Mn-SOD in NGF signaling, extending the above observations to a
non-neuronal cell background. These data are therefore consistent with
the idea that elimination of harmful oxygen species from mitochondria
through the transcriptional up-regulation of Mn-SOD plays a critical
role in NGF-dependent cytoprotection (Fig.
8).
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Fig. 7.
Involvement of Mn-SOD in
NGF-dependent cell survival. A, PC12 cell
rescue by NGF is reduced by an antisense construct for Mn-SOD.
a, cells were infected with a Mn-SOD antisense construct
(Mn-SOD AS) or the corresponding empty vector (pLPC) and
selected in 2.5 µg/ml puromycin as previously described. Cell
survival was assessed by flow cytometry 48 h after serum
withdrawal. Values are the mean ± S.D. of duplicate samples.
b, steady-state Mn-SOD content in mock and
Mn-SOD/AS-transduced cells was evaluated at the end of selection,
immediately before serum withdrawal. B,
Trk-dependent survival signaling in transformed fibroblasts
requires Mn-SOD. a, SOD2 +/+- and SOD2 /
-transformed
fibroblasts expressing the rat Trk receptor were grown in the indicated
media, and cell survival was determined by flow cytometry (propidium
iodide exclusion) after 48 h of incubation. NGF significantly
reduced cell death in the serum-free medium of wild-type but not of
SOD2-deficient cells. Values are the mean ± S.D. of duplicate
samples. b, anti-Mn-SOD Western blot analysis of total
protein lysates from SOD2 +/+/Trk and SOD2
/
/Trk cells. The protein
band corresponding to SOD2 is completely absent in the SOD
/
sample. c, NGF-dependent phosphorylation
(p) of CREB in SOD2 +/+ and SOD2
/
MEFs. Early NGF
signaling is preserved in SOD2 knock-out cells. C,
control.
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Fig. 8.
A model for the dual role of ROS in PC12 cell
rescue by NGF. The model outlines the dual role of ROS in survival
signaling and in cell death. Oxygen species that are rapidly generated
in the cell cytosol in response to NGF and Rac-1 have a role in
survival signaling to Akt and CREB, whereas harmful mitochondrial
oxidants produced at the onset of apoptotic death are reduced by the
neurotrophin, likely through the induction of the mitochondrial
scavenger Mn-SOD.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1258 of the human Mn-SOD promoter (28), and
overexpression of CREB leads to Mn-SOD overexpression in PC12 and 293T
cells (Fig. 7). Moreover, cell treatment with NAC drastically decreases
Mn-SOD expression in response to NGF concomitantly with a significant
reduction in the phosphorylation/activation of CREB. Interestingly,
Mn-SOD is induced by a constitutively active mutant of CREB in a
ROS-independent fashion, suggesting that this factor is a downstream
target for redox signaling in the cascade linking NGF to the
up-regulation of Mn-SOD. Further studies utilizing CREB inhibitory
molecules will be required to assess the relative role of this factor
in the induction of Mn-SOD by NGF with respect to other potential
candidates such as nuclear factor
B. However, even though the
mechanisms involved are unclear, the evidence of Mn-SOD induction
through a ROS-dependent cascade is biologically sound and
in line with other findings that oxidants are inducers of antioxidant
enzymes in both prokaryotic (52) and eukaryotic cells (53).
(56) and is supported by data displayed in Fig. 6, which shows the
opposite effect of a generic (NAC) versus a mitochondrially targeted (Mito Q) antioxidant in cell response to serum deprivation and
to NGF. Although Mito Q well mimics the compartment-specific effect of
Mn-SOD, NAC freely diffuses throughout the cell, inhibiting survival
signaling. On the other hand, because NAC can also remove harmful
oxidants from mitochondria, this effect could partially mitigate the
upstream inactivation of ROS-dependent antiapoptotic signals, thereby explaining the discrepancy between the profound inhibition of Akt, CREB, and Mn-SOD and the limited decrease in cell
survival observed in cells treated with NAC (compare Figs. 3 and 6).
One final consideration deals with the significance of the PC12 model
with respect to the general mechanisms of neuroprotection by NGF.
Although NGF plays a key role in target-determined cell survival within
the developing nervous system (4), the effect of the neurotrophin on
undifferentiated, serum-starved PC12 cells depicts a more general
situation of growth factor-dependent cell rescue from an
acute insult. However, although the above observations need to be
confirmed in vivo or in different cellular models more representative of the physiological role of NGF, it should be noted
that a role for neurotrophins in adult cell recovery from damage
("cell rescue") has also been proposed (4). Notwithstanding these
limitations, the presented observations on redox-dependent regulation of NGF signaling and on NGF induction of Mn-SOD may contribute to understanding the molecular mechanisms of neuronal protection by neurotrophins and clarification of the intricate connections between oxidative stress, antioxidant enzymes, and neuropathology.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. A. Bellacosa, D. D. Ginty, A. Hall, S. Lowe, and D. Mercanti for generously providing reagents and expression constructs and students Salvatore Fusco and Daniela Ferraro for experimental contributions.
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FOOTNOTES |
---|
* This work was supported by Consiglio Nazionale delle Ricerche, Target project on Biotechnology Grant 01.00718.PF49, and by Ministero della Universita' e Ricerca Scientifica e Technologica/Consiglio Nazionale delle Ricerche Biotechnology Program L.95/95 Grant 01.00184.PF31.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.
This paper is dedicated to the memory of Eraldo Antonini, eminent biochemist, prematurely deceased 20 years ago on March 19, 1983.
§ Both authors contributed equally to this work.
To whom correspondence should be addressed: Institute of
General Pathology, Largo F. Vito 1, 00168 Rome, Italy. Tel.:
39-06-30154914; Fax: 39-06-3386446; E-mail:
Tgaleotti@rm.unicatt.it.
Published, JBC Papers in Press, February 27, 2003, DOI 10.1074/jbc.M301089200
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ABBREVIATIONS |
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The abbreviations used are: ROS, reactive oxygen species; Mn-SOD, manganese-dependent superoxide dismutase; CuZn-SOD, copper zinc-dependent superoxide dismutase; DCF-DA, dichlorofluorescin diacetate; DHE, dihydroethidium; FCS, fetal calf serum; 2-ME, 2-methoxyestradiol; MEF, mouse embryonic fibroblast; Mito Q, mitoquinol/mitoquinone; NAC, N-acetyl-L-cysteine; NGF, nerve growth factor; FKHR, Forkhead-related transcription factor; CREB, cAMP-responsive element-binding protein; PI, phosphatidylinositol; GFP, green fluorescent protein.
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