From the Instituto de Investigaciones
Biomédicas and the Departamento de Bioquímica, Facultad
de Medicina, Universidad Autónoma de Madrid, 28029 Madrid, Spain,
the ¶ Departamento de Bioquímica, Facultad de Medicina,
Universidad de las Palmas de Gran Canaria, 35016 Gran Canaria,
Spain, and the ** Department of Pharmacology, New York
Medical College, Valhalla, New York 10595
Received for publication, September 6, 2002, and in revised form, February 7, 2003
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ABSTRACT |
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The survival signal elicited by the
phosphatidylinositol 3-kinase (PI3K)/Akt1 pathway has been correlated
with inactivation of pro-apoptotic proteins and attenuation of the
general stress-induced increase in reactive oxygen species (ROS).
However, the mechanisms by which this pathway regulates intracellular
ROS levels remain largely unexplored. In this study, we demonstrate
that nerve growth factor (NGF) prevents the accumulation of ROS in
dopaminergic PC12 cells challenged with the Parkinson's
disease-related neurotoxin 6-hydroxydopamine (6-OHDA) by a mechanism
that involves PI3K/Akt-dependent induction of the stress
response protein heme oxygenase-1 (HO-1). The effect of NGF was
mimicked by induction of HO-1 expression with
CoCl2; by treatment with bilirubin, an end
product of heme catabolism; and by infection with a retroviral
expression vector for human HO-1. The relevance of HO-1 in
NGF-induced ROS reduction was further demonstrated by the evidence that
cells treated with the HO-1 inhibitor tin-protoporphyrin or infected
with a retroviral expression vector for antisense HO-1 exhibited
enhanced ROS release in response to 6-OHDA, despite the presence of the
neurotrophin. Inhibition of PI3K prevented NGF induction of HO-1
mRNA and protein and partially reversed its protective effect
against 6-OHDA-induced ROS release. By contrast, cells transfected with
a membrane-targeted active version of Akt1 exhibited increased HO-1
expression, even in the absence of NGF, and displayed a greatly
attenuated production of ROS and apoptosis in response to 6-OHDA. These
observations indicate that the PI3K/Akt pathway controls the
intracellular levels of ROS by regulating the expression of the
antioxidant enzyme HO-1.
High levels of reactive oxygen species
(ROS)1 induce cell death
in the nervous system and have been associated with a number of
pathologies such as Parkinson's disease (1). Neurotrophins, including
nerve growth factor (NGF), promote survival of target neurons and
attenuate ROS-induced cell death (2). A prominent mechanism involved in
NGF-induced cell survival consists of the activation of
phosphatidylinositol 3-kinase (PI3K) and its downstream effectors,
including the protein kinase B/Akt family of Ser/Thr kinases
(3-5). This pathway exerts protective actions against oxidative damage
in central and peripheral neurons (6). In this context, neuregulin
prevents H2O2 induction of ROS in a
PI3K-dependent manner (7). Loss of oxidative stress
tolerance with aging has been linked in part to reduced Akt kinase
activity in old rats (8). Concerning neurodegeneration, we have
recently reported the protective effect of active Akt1 against peptides
of However, although those studies support an involvement of Akt in
counteracting oxidative damage in neurons, a direct role of this
pathway in the regulation of canonical antioxidant defenses, represented by the superoxide dismutases and/or the
catalase/glutathione peroxidase system, has not been identified. On the
contrary, recent reports on the dauer stage of
Caenorhabditis elegans and quiescent mammalian cells
indicate that, in the absence of active PI3K and Akt, forkhead
transcription factors (DAF-16 and FOXO3a, respectively) increase the
expression of mitochondrial superoxide dismutase (12, 13). Moreover,
mutations in the daf-2 network of C. elegans that
inactivate PI3K and Akt lead to up-regulation of a cytosolic catalase
(14); and in Drosophila melanogaster, inactivation of this
pathway results in increased superoxide dismutase activity (15).
Therefore, activation of PI3K and Akt must lead to up-regulation of
other ROS detoxification systems.
In addition to the well characterized ROS scavenger systems mentioned
above, emerging evidence supports a role for heme oxygenase (HO)
enzymes as important components of the cellular antioxidant armamentarium (16, 17). The heme oxygenase family is composed of at
least two well characterized isoenzymes: inducible HO-1 and
constitutive HO-2. HO-1, also known as HSP32 (heat
shock protein of 32 kDa), is
a stress response protein whose expression is induced in practically
all tissues and cells tested in response to multiple oxidative insults
such as heme, UV light, heavy metals, glutathione depletion, and
H2O2. This enzyme catalyzes the stepwise
degradation of heme to release free iron and equimolar concentrations
of carbon monoxide (CO) and the linear tetrapyrrol biliverdin, which is converted to bilirubin by the enzyme biliverdin reductase. Many reports
have established the potent antioxidant activity of biliverdin and
bilirubin and the cytoprotective actions of CO on vascular endothelium
and nerve cells (16-19). Therefore, it is now widely accepted that
induction of heme catabolism represents an adaptive, and ultimately
protective, response to oxidative injury. Of particular relevance to
this study, it has also been shown that the normally low levels of HO-1
expression in neurons (20, 21) dramatically increase after formation of
brain neurofibrillary tangles in Alzheimer's patients (22) and
neuronal Lewy bodies in the substantia nigra of Parkinson's patients
(23) and that cerebellar granular neurons overexpressing HO-1 are
resistant to glutamate-mediated oxidative stress (24).
The progressive deterioration of catecholaminergic cells in
Parkinson's patients has been attributed, at least in part, to the
high vulnerability of these cells to oxidative damage. One potential
source of ROS in the substantia nigra includes the autoxidation of the
neurotransmitter dopamine to generate 6-hydroxydopamine (6-OHDA) (25).
In fact, 6-OHDA is present in rodent and human brains and has been
widely used in experimental models of Parkinson's disease. Evidences
derived from in vivo and in vitro experimental models of this disease have demonstrated that 6-OHDA neurotoxicity involves oxidative damage to catecholaminergic neurons (26) via the
generation of hydroxyl radicals, monoamine oxidase-mediated formation
of H2O2, and mitochondrial generation of
superoxide (25).
With the aim to determine whether PI3K and Akt modulate the heme
oxygenase system of ROS detoxification, we analyzed the effect of NGF
on the regulation of HO-1 expression in catecholaminergic PC12 cells.
We show that NGF-induced activation of PI3K/Akt up-regulates the
expression and activity of HO-1, which, in turn, provides protection
against 6-OHDA-induced oxidative damage in PC12 cells. These data
contribute to revealing the mechanism whereby NGF and PI3K/Akt provide
protection against oxidative injury and may be potentially relevant in
the development of new therapies for neurodegenerative disorders such
as Parkinson's disease.
Cell Culture, Transfections, and Reagents--
PC12 cells (a
gift of Dr. H. Kleinman, NIDCR, National Institutes of Health,
Bethesda, MD) were grown in Dulbecco's modified Eagle's medium
supplemented with 7.5% fetal bovine serum, 7.5% heat-inactivated
horse serum, and 80 µg/ml gentamycin. Eukaryotic expression vectors
for enhanced green fluorescent protein (EGFP) and myristoylated
(myr)-EGFP-Akt1 have been described elsewhere (13). Stable transfection
of PC12 cells was performed with Superfect transfection reagent (QIAGEN
Inc., Valencia, CA) according to the manufacturer's instructions. The
amphotropic retroviral packaging cell lines PT67 and PA317
(Clontech) were used to collect
replication-deficient retroviruses containing the retroviral vector
(LXSN) expressing human HO-1
antisense cDNA under the control of the
human HO-1 promoter (LSN-HOP-HHO-1-AS) and
human HO-1 in the sense orientation (LSN-HHO-1), respectively. These
retroviral constructs have been described elsewhere (27). PC12 cells
infected with the pLNCX retroviral vector (a generous gift of Dr. R. Perona, Instituto de Investigaciones Biomédicas,
Universidad Autónoma de Madrid) were used as controls for
HO-1 infections. Transfected and infected cells were selected in 0.5 mg/ml G418. The reagents employed were NGF (Peprotec, Rocky
Hill, NJ), tin-protoporphyrin (Protoporphyrin Products, Logan, UT),
and 6-OHDA, actinomycin D, cycloheximide, LY294002, hemin,
bilirubin, and CoCl2 (Sigma).
HO Assays--
Microsomal HO-1 activity was determined
spectrophotometrically in PC12 cell microsomes incubated for 1 h
in the dark at 37 °C in the presence of hemin (10 µM),
NADPH (20 µM), and 1 mg of protein from rat liver cytosol
as a source of biliverdin reductase (28). Reactions were terminated by
adding 1 ml of chloroform, and the concentration of bilirubin was
determined from the difference in absorbance between 464 and 530 nm
using an extinction coefficient of 40 mM -1
cm Analysis of mRNA Levels by Reverse
Transcriptase-PCR--
Total cellular RNA was extracted using TRIzol
reagent (Invitrogen) (29). Equal amounts (1 µg) of RNA from the
different treatments were reversed-transcribed (75 min, 42 °C) using
Superscript II reverse transcriptase (Invitrogen). Amplification of
cDNA was performed in 25 µl of PCR buffer (10 mM
Tris-HCl, 50 mM KCl, 5 mM MgCl2,
and 0.1% Triton X-100, pH 9.0) containing 2.5 mM
digoxigenin-dUTP, 0.6 units of Taq DNA polymerase, and 30 pmol of synthetic gene-specific primers for HO-1 (forward,
5'-AAGGCTTTAAGCTGGTGATGG-3'; and reverse, 5'-AGCGGTGTCTGGGATGAACTA-3').
To ensure that equal amounts of reverse-transcribed RNA were added to
the PCR, the glyceraldehyde-3-phosphate dehydrogenase or Immunoblotting--
Primary anti-HO-1 antibodies were purchased
from Stressgen and Oxys. Anti-protein-disulfide isomerase
antibodies were a gift from Dr. J. G. Castaño
(Instituto de Investigaciones Biomédicas, Universidad
Autónoma de Madrid-Consejo Superior de Investigaciones Científicas). Cells were grown in 60-cm plates, washed once
with cold phosphate-buffered saline, and lysed on ice with 200 µl of lysis buffer (1% Nonidet P-40, 10% glycerol, 137 mM NaCl,
20 mM Tris-HCl, pH 7.5, 1 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 20 mM NaF, 1 mM sodium pyrophosphate, and 1 mM
Na3VO4). Lysates were precleared by
centrifugation, resolved by SDS-PAGE, and transferred to Immobilon-P
membranes (Millipore Corp., Madrid, Spain). Blots were analyzed with
anti-HO-1 antibodies (1:1000). Appropriate peroxidase-conjugated
secondary antibodies (1:10,000) were used to detect the proteins of
interest by enhanced chemiluminescence. Relative protein levels were
determined by scanning densitometry and analysis with NIH Image software.
Confocal Microscopy--
Cells were seeded on glass-bottom 35-mm
plates (Willco, Amsterdam, The Netherlands). Following overnight
serum starvation, cells were incubated with 40 µM 6-OHDA
for 6 h or with 1 mM H2O2 for
45 min and afterward with 2 µM hydroethidine (HE)
(Molecular Probes, Inc., Eugene, OR) for 1 h at 37 °C. Confocal
microscopy was performed using a Leica SP2 system. For HE, we used an
excitation wavelength of 488 nm, and fluorescence was detected at
wavelengths between 557 and 740 nm. For Hoechst 33258, we used an
excitation wavelength of 351 nm, and fluorescence was detected at
wavelengths between 386 and 512 nm.
Flow Cytometry--
A FACScan flow cytometer (BD Biosciences,
Madrid) was used to analyze fluorescence from HE (band pass
575/24 nm), annexin V-phycoerythrin (PE) (band pass 620/22 nm), and
7-aminoactinomycin D (7-AAD) (band pass 575/24 nm). Intracellular ROS
were detected with HE. Cells were detached from the plates, washed with
phosphate-buffered saline, incubated with 2 µM HE for
1 h at 37 °C, and analyzed immediately. Base-line incorporation
of the probe was determined in serum-starved cells incubated in 2 µM HE at 4 °C. Variations in HE fluorescence between
experiments were attributed to slight differences in the
photomultiplier parameters used during data acquisition and to small
differences among batches of HE. For apoptosis assays, floating and
attached cells were transferred to conical tubes and spun at 300 × g for 10 min. Cells were resuspended in annexin V binding
buffer (10 mM HEPES-NaOH, pH 7.4, 140 mM NaCl,
and 2.5 mM CaCl2) at 106
cells/ml. Cells were incubated for 15 min at 25 °C in a 1:20 solution of annexin V-PE (Pharmingen, Madrid) supplemented with 1 µg/ml 7-AAD (Sigma) and analyzed immediately.
Statistics--
Student's t test was used to assess
differences between groups. A p value of <0.05 was
considered significant. Unless indicated, all experiments were
performed al least three times with similar results. The values in
graphs correspond to the average of at least three samples. Bars
indicate S.D.
6-OHDA Induces ROS Generation in PC12 Cells--
To assess
variations in ROS production, we used the ROS-sensitive fluorescent
probe HE, a well established sensor of oxidative stress, most specific
of superoxide anion (10, 30, 31). Mitochondria are the primary sites of
HE oxidation; and once converted to ethidine, this product
accumulates intercalated into nucleic acids at the nucleus (31).
Serum-starved PC12 cells were treated with 40 µM 6-OHDA
for 6 h or with 1 mM H2O2 for
45 min as a control of oxidative stress, and 2 µM HE was
added for 1 h. As shown in Fig.
1A, counterstaining of these
cells with the nuclear dye Hoechst 33258 and analysis with confocal
microscopy evidenced HE accumulation mostly in the nuclei of cells
submitted to oxidative damage. We then quantified the incorporation of
HE in 6-OHDA-treated cells by flow cytometry as shown in Fig.
1B. Untreated cells exhibited weak HE incorporation,
indicating that the basal amount of ROS in these cells is low. By
contrast, 6-OHDA induced a dose-dependent incorporation of
the probe. Similar results were obtained with the green-emitting
fluorophore 2',7'-dichlorodihydrofluorescein diacetate (data not
shown). However, because heme and hemoproteins may interfere with the
oxidation of this dye in a ROS-independent manner (32), we concentrated
the rest our work on HE.
Next, we analyzed the effect of NGF on prevention of 6-OHDA-induced
release of ROS. As shown in Fig.
2A, a 16-h pretreatment with
20 ng/ml NGF significantly reduced the 6-OHDA-induced incorporation of
HE, indicating that NGF attenuates ROS production. Moreover, as shown
in Fig. 2B, this effect was dependent on the length of NGF
pretreatment. Thus, attenuation of ROS release by NGF required the
presence of this neurotrophin at least 3 h prior to the addition of 6-OHDA. These results suggest that NGF induces the expression of a
gene(s) essential to ROS antagonism.
NGF Up-regulates HO-1 Expression--
Considering that HO-1 is an
inducible enzyme with strong antioxidant properties, we studied the
effect of NGF on the expression of this protein. Treatment of PC12
cells for 6 h with NGF resulted in a 2-3-fold increase in HO
activity (Fig. 3A). This
increase was sensitive to pretreatment with actinomycin D or
cycloheximide, suggesting that NGF enhances the expression of the
inducible heme oxygenase isoform, HO-1. Indeed, as shown in Fig. 3
(B and C), the increase in HO activity correlated
with increased levels of HO-1 mRNA and protein. Moreover, the
increase in mRNA was also sensitive to cycloheximide, suggesting,
in agreement with other studies (33, 34), that induction of HO-1
transcription involves de novo protein synthesis. Moreover,
as shown in Fig. 3D, NGF markedly elevated HO-1 mRNA,
reaching a maximum within 3 h of treatment and declining by
9 h of NGF exposure. In addition, induction of HO-1 protein by NGF
was delayed (Fig. 3E). There was a slight increase observed
at 1.5-3 h, with a maximum 6-8-fold elevation at 9 h; but HO-1
protein levels remained elevated for at least for 12 h after NGF
stimulation. NGF failed to stimulate HO-2 expression (data not shown),
further indicating that this neurotrophic factor specifically
up-regulates the HO-1 isoform.
Induction of HO-1 Expression and Exogenous Addition of Bilirubin
Attenuate ROS Generation by 6-OHDA--
To assess the relevance of
HO-1 in 6-OHDA detoxification, we treated PC12 cells with
CoCl2, a well established inducer of HO-1 expression that
promotes depletion of the intracellular GSH pool and does not appear to
significantly alter the activity of other antioxidant enzymes,
including superoxide dismutase, catalase, and glutathione peroxidase
(35). Heme oxygenase expression was induced in PC12 cells following
incubation with CoCl2 for 12 h as indicated in Fig.
4A. Plateau induction of HO-1
protein was observed with ~100 µM CoCl2.
Contrary to hemin, another commonly used inducer of HO-1,
CoCl2 did not display significant toxicity, at least for
the length of these experiments (data not shown). Following 12 h
of CoCl2 induction of HO-1, cells were treated with 6-OHDA
as indicated in Fig. 4B. CoCl2 pretreatment
strongly attenuated the production of ROS in the presence of 6-OHDA. To further confirm that the CoCl2 protection was due to
induction of HO-1, we analyzed the protective effect of bilirubin.
Serum-starved cells were pretreated with 10 µM bilirubin
for 12 h prior to the treatment with 6-OHDA. As shown in Fig.
4B, bilirubin also significantly attenuated the 6-OHDA
induction of ROS, although to a lesser extent compared with
CoCl2. These results indicate that induction of HO-1
prevents ROS production by 6-OHDA.
Moderate Overexpression of HO-1 Attenuates 6-OHDA-induced ROS
Production--
We also overexpressed HO-1 in PC12 cells and
determined its effect on 6-OHDA-induced HE fluorescence. PC12 cells
were infected with the retroviral expression vector LSN-HHO-1, which
has been described previously (27). We could not find cell clones with levels of HO-1 overexpression higher than 2-3-fold, suggesting that
higher constitutive levels of HO activity may be deleterious (see
"Discussion"), probably as a result of the depletion of
intracellular heme reservoirs. A pool of five clones overexpressing
HO-1 by 2.3 ± 0.3-fold (Fig. 4C) was used to assess
the relevance of this enzyme in the 6-OHDA-induced production of ROS.
Although modest, comparative levels of overexpression conferred
protection against glutamate-mediated oxidative stress in neurons of
transgenic mice (24). As shown in Fig. 4D,
HO-1-overexpressing cells exhibited a 6-OHDA-induced HE incorporation
that was intermediate between that induced by the toxin in control
untreated and NGF-treated cells. These results further suggest an
antioxidant effect of HO-1. Interestingly, NGF produced a similar
attenuation of ROS in both control and HO-1-overexpressing cells in the
presence of 6-OHDA, suggesting that the levels of HO-1 induced by the
neurotrophin may be already at saturating doses. An additional
interpretation might be that NGF induces other antioxidant genes or
pathways that, in concert with HO-1, facilitate maximum protection
against 6-OHDA-induced ROS (see "Discussion").
NGF Does Not Prevent 6-OHDA-generated ROS in HO-1-depleted
Cells--
To determine the relevance of HO-1 induction in the NGF
protection against oxidative stress, we analyzed the effect of
pharmacological inhibition of heme oxygenase with tin-protoporphyrin, a
competitive inhibitor of HO-1. PC12 cells were treated with 20 ng/ml
NGF and various tin-protoporphyrin concentrations in combination as
indicated in Fig. 5A.
Tin-protoporphyrin alone dose-dependently increased HE
incorporation, indicating that inhibition of heme oxygenase increases
cellular ROS levels. Interestingly, NGF could not prevent ROS
generation in the presence of tin-protoporphyrin either in untreated
cells or in cells treated with 6-OHDA, particularly at the highest
doses.
We also analyzed the 6-OHDA-induced HE incorporation in cells
retrovirally transduced with antisense HO-1 constructs. The antisense
retroviral expression vector for HO-1 under the control of its own
promoter (LSN-HOP-HHO-1-AS) has been described previously (27). In
agreement with previous reports (36), cells transfected with this
antisense constructs grew more slowly (data not shown), suggesting
abnormalities in cell cycle progression. Fig. 5B shows the
endogenous levels of HO-1 in control PC12 cells and in two clones (AS1
and AS2) selected after retroviral transduction with LSN-HOP-HHO-1-AS. Both cell clones exhibited lower HO-1 protein levels compared with control cells (54 ± 8 and 46 ± 12% of
the control for AS1 and AS2, respectively). Moreover, hemin yielded lower HO-1 protein levels in both antisense clones, indicating that
HO-1 expression is impaired in these cells. We then analyzed the levels
of 6-OHDA-induced incorporation of HE as shown in Fig. 5C.
Treatment with 40 µM 6-OHDA induced a significantly
higher incorporation of HE in the antisense clones compared with
control cells, indicative of stronger production of ROS in the
HO-1-depleted cells. Interestingly, pretreatment with NGF for 16 h
did not prevent 6-OHDA induction of ROS in either of the antisense
HO-1-transduced cells. These observations suggest that induction of
HO-1 is essential for NGF to attenuate 6-OHDA-induced production of
ROS; and because both antisense clones still harbor a fraction of HO-1
protein, it is likely that cells require a threshold amount of HO-1,
above that present in these cells, to be efficiently protected against this neurotoxin. Taken together, these results further support a role
for HO-1 in the NGF-induced antioxidant response.
NGF Induces HO-1 Expression in a PI3K- and
Akt-dependent Manner--
Next, we investigated the
contribution of the NGF-activated PI3K/Akt survival pathway to the
induction of HO-1 expression. For this purpose, PC12 cells were
transfected with either a control expression vector for EGFP or a
membrane-targeted active fusion of EGFP and Akt1 (myr-EGFP-Akt1), which
has been described previously (13). The main advantage of using these
constructs is to select transfected PC12 cells by preparative cell
sorting. Serum-starved control EGFP cells were pretreated for 15 min
with 40 µM LY294002, an inhibitor of PI3K, and then
stimulated with 20 ng/ml NGF for 6 h. As shown in Fig.
6, the PI3K inhibitor significantly
blocked the NGF induction of both HO-1 mRNA (Fig. 6A)
and protein (Fig. 6B) in control EGFP cells. Interestingly,
myr-EGFP-Akt1 cells exhibited higher basal levels of HO-1 mRNA
(Fig. 6A) and protein (Fig. 6C) compared with
control EGFP cells. Moreover, the inhibition of PI3K in myr-EGFP-Akt1
cells produced only a small decrease in HO-1 mRNA (Fig.
6A), suggesting that myristoylated Akt1 kinase, which does
not require 3D'-phosphoinositides for membrane anchorage, is sufficient to activate HO-1 expression. Similar results were obtained when cells were treated with 100 nM wortmannin,
another PI3K inhibitor without structural homology to LY294002 (data
not shown). Taken together, these results demonstrate that active Akt
induces the expression of HO-1 and that NGF regulates the expression of
this enzyme, at least in part, through the PI3K/Akt pathway.
The PI3K/Akt Pathway Is Necessary and Sufficient to
Attenuate 6-OHDA Generation of ROS--
We then analyzed the effect of
the PI3K/Akt pathway on the attenuation of 6-OHDA-induced oxidative
stress. Serum-starved PC12 cells were pretreated for 15 min with 40 µM LY294002 prior to the addition of NGF. Following a
16-h incubation with NGF, cells were treated with 6-OHDA for 6 h.
As shown in Fig. 7A, LY294002 reversed the protective effect of NGF against 6-OHDA-induced ROS, further supporting the concept that the PI3K survival pathway controls
ROS levels, at least in part, by inducing HO-1 expression. We also
analyzed 6-OHDA-induced ROS production in myr-EGFP-Akt1 cells, which
harbored higher HO-1 levels compared with control EGFP cells (Fig. 6).
As shown in Fig. 7B, HE incorporation was fully prevented in
myr-EGFP-Akt1 cells at low 6-OHDA concentrations (20 µM)
compared with control EGFP cells. At higher concentrations (40 µM), myr-EGFP-Akt1 cells still exhibited a strong
attenuation of HE staining. In conclusion, active Akt protects PC12
cells against 6-OHDA-induced oxidative stress, at least in part, by activating the expression of the antioxidant enzyme HO-1.
Active Akt Prevents 6-OHDA-induced Apoptosis--
Finally, we
investigated whether the deregulated and active form of Akt might
attenuate the apoptotic effect of 6-OHDA. We analyzed the loss of
plasma membrane asymmetry, which characterizes early apoptosis and
results in the exposure of phosphatidylserine to the outer plasma
membrane leaflet. This alteration was determined by staining with the
phosphatidylserine-binding protein annexin V conjugated to PE. To
discriminate early apoptosis from late apoptosis and necrosis, cells
were simultaneously stained with 7-AAD, which stains only cells lacking
membrane integrity. Serum-starved control EGFP and myr-EGFP-Akt1 cells
were submitted to 6 h of incubation with 40 µM
6-OHDA and then stained with annexin V-PE (1:20, v/v) and 2 µM 7-AAD prior to flow cytometric analysis. As shown in
Fig. 8A, most untreated EGFP
and myr-EGFP-Akt1 cells were annexin V-PE- and 7-AAD-negative,
indicating that they were viable. However, as shown in Fig.
8B, after a 6-h treatment with 40 µM 6-OHDA,
~12% of the EGFP cells were annexin V-PE-positive and
7-AAD-negative, indicating that this population was undergoing early
apoptosis. By contrast, the fraction of myr-EGFP-Akt1 cells that were
annexin V-PE-positive and 7-AAD-negative after the 6-OHDA insult did
not increase significantly. These results indicate that, in addition to
attenuation of 6-OHDA-induced oxidative stress, the Akt survival
pathway also protects against 6-OHDA-induced apoptosis.
This study documents a new physiological role for the PI3K/Akt
survival pathway activated by the neurotrophic factor NGF: control of
intracellular levels of oxygen free radicals by regulating the
expression of HO-1. Previous studies have indicated that NGF elicits a
protective effect against oxidative stress both in PC12 cells and in
cultured neurons (37, 38). It has also been shown that NGF may modulate
the canonical antioxidant machinery of ROS detoxification by increasing
the expression of catalase and glutathione peroxidase activities (39,
40). Our experiments further demonstrate that NGF regulates the
expression of the novel antioxidant mechanism involving the stress
protein HO-1. Moreover, enhanced HO-1 expression is essential for NGF
function in ROS detoxification because cells expressing antisense HO-1
retroviral constructs were insensitive to NGF protection against
6-OHDA-induced oxidative stress. Although PI3K/Akt is a well documented
pathway involved in protecting against apoptosis insults, including
oxidative stress, this is the first report linking this survival
pathway with a specific enzyme involved in ROS detoxification of
mammalian cells. We show that the PI3K/Akt pathway is both necessary
and sufficient for NGF-dependent abrogation of ROS levels
in PC12 cells exposed to 6-OHDA.
The protective role of the stress protein HO-1 in the brain has not
been accepted until recently. Initially, post-mortem examination of
human brain specimens revealed that expression of HO-1 immunoreactivity decorates Lewy bodies in the substantia nigra of Parkinson's patients (23). This led to the assumption that HO-1 overactivity might not
protect, but rather contribute to the development of parkinsonism because iron accumulation in dopaminergic neurons may contribute to
chronic oxidative damage, leading to neurodegeneration in Parkinson's patients. A protective role for HO catabolism in the nervous system has
been also challenged by the fact that hyperbilirubinemia is commonly
associated with nerve cell injury and brain damage during severe
neonatal jaundice and Crigler-Najjar type II syndrome (41, 42).
Consistent with these observations, we could not obtain high
constitutive levels of HO-1 expression either in HO-1-overexpressing cells (2.3-fold) or in myr-EGFP-Akt1 cells (2.5-fold). It is
interesting, however, that short-term stimulation with NGF or other
inducers of HO-1 such as hemin and CoCl2 may yield much
higher transient levels of HO-1 expression. These observations are in
agreement with the notion that HO-1 is a stress response protein (HSP32).
Therefore, a key point toward establishing the protective or noxious
effect on the HO system under conditions of oxidative injury may be
related to the relative abundance of HO enzymes and their products. Low
concentrations of bilirubin derived from HO-2 overexpression protect
against neuronal oxidant injury (43), and moderated levels of bilirubin
exert antioxidant actions in the neonate and protect against
retinopathy in premature newborns (44). Likewise, recent reports have
shown that transgenic mice overexpressing moderate levels of HO-1
display augmented resistance to neural damage in animal models of
cerebral ischemia (45) or after H2O2- and
glutamate-induced oxidant stress in vitro (24). Accordingly,
we have shown that constitutive but moderate overexpression of HO-1
attenuated 6-OHDA-induced ROS generation.
The HO-1 gene contains a complex promoter with a large variety of
regulatory elements (46). Some of these sequences might be putative
candidates for regulation by PI3K and Akt. As a heat shock protein, the
promoter of HO-1 contains several heat shock elements that may be
negatively regulated by glycogen synthase kinase-3-mediated
phosphorylation of heat shock factor-1. Because Akt phosphorylates and
inhibits glycogen synthase kinase-3 (47), these might be sites of
indirect regulation by this pathway. Another putative site involves the
antioxidant-responsive element. During oxidative stress, the basic
leucine zipper transcription factor Nrf2 heterodimerizes with
small Maf to bind and activate antioxidant-responsive element sequences
(48). Although the putative regulation of Nrf2 by PI3K is poorly
defined, recent reports suggest that PI3K regulates nuclear
translocation of Nrf2 through actin rearrangement in response to
oxidative stress (49). We are currently analyzing these and other
promoter regions that might ultimately respond to PI3K/Akt regulation.
Given the variety of enzymes involved in oxidative detoxification, a
striking finding of this study is the strong dependence on HO-1 to
protect against 6-OHDA-induced oxidative stress. This may be explained
by considering the mechanism involved in 6-OHDA toxicity. Probably,
H2O2 is the most abundant form of ROS generated by 6-OHDA (25), and bilirubin is particularly well suited to deal with
this compound because nanomolar amounts of bilirubin reduce micromolar
amounts of H2O2 (43). Moreover, other
antioxidant enzymes may be regulated by by-products of HO-1 activity,
thus contributing to ROS detoxification. For example, HO-1 activates the expression of mitochondrial superoxide dismutase in neonatal rat
astroglia challenged with dopamine (50).
Finally, we discovered that membrane-targeted active Akt prevented the
effects of 6-OHDA not only on ROS production, but also on apoptosis,
further suggesting that moderate overexpression of HO-1 through the
PI3K/Akt survival pathway may be an important element for prevention of
both phenomena. Considering the importance of apoptosis and ROS in
neurodegeneration, this study suggests that activation of the PI3K/Akt
pathway might have a therapeutic use in the treatment of oxidative
stress-related neurodegenerative disorders such as Parkinson's disease.
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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-amyloid protein, characteristic of senile plaques found in the
brains of Alzheimer's patients (9); against the Parkinson's
disease-inducing toxin 1-methyl-4-phenylpyridinium (10); and against
apoptotic concentrations of H2O2 (11). In these
cases, expression of a constitutively active version of Akt prevented
the increase in ROS that follows treatment of PC12 cells with these toxins.
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1.
-actin
housekeeping gene was amplified using commercially available
oligonucleotides (Clontech). After an initial denaturation step (4 min, 94 °C), amplification of each cDNA was performed for 17, 19, 21, 23, and 25 cycles (HO-1,
glyceraldehyde-3-phosphate dehydrogenase, or
-actin) using a thermal
profile of 1 min at 94 °C (denaturation), 1 min at 58 °C
(annealing), and 1 min at 72 °C (elongation). The optimal number of
cycles within the linear range of amplification were selected as 23 for
HO-1 and 19 for glyceraldehyde-3-phosphate dehydrogenase or
-actin
(data not shown). The amplified PCR products were resolved by 1.8%
agarose gel electrophoresis and transferred to nylon membranes.
Chemiluminescence detection was performed with a digoxigenin
luminescence detection kit (Roche Molecular Biochemicals, Basel, Switzerland).
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Fig. 1.
6-OHDA induces a rise in ROS in PC12 cells as
determined by HE staining. A, confocal
microscopy pictures of representative fields from untreated and 6-OHDA-
and H2O2-treated cells stained with HE and
Hoechst 33258. Serum-starved PC12 cells were treated with 40 µM 6-OHDA for 6 h or with 1 mM
H2O2 for 45 min, and 2 µM HE was
added during the last hour of the experiment. B,
quantitative determination by flow cytometry of 6-OHDA-induced
oxidative stress by HE staining. Cells were treated as described for
A with the indicated concentrations of 6-OHDA. A
representative sample of 10,000 cells is shown for untreated and 20 and
40 µM 6-OHDA-treated cells.
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Fig. 2.
NGF antagonizes the oxidant effect of
6-OHDA. A, NGF prevents 6-OHDA induction of
ROS. Serum-starved cells were treated with 20 ng/ml NGF for 16 h,
and 6-OHDA and HE were added for the last 6 and 1 h, respectively.
B, prevention of 6-OHDA-induced ROS by NGF depends on the
length of the NGF pretreatment. Serum-starved cells were incubated with
20 ng/ml NGF for the indicated times. They were then treated with 40 µM 6-OHDA for 6 h and with HE for 1 h.
Asterisks denote p < 0.001, NGF-treated
versus untreated groups. a.u., absorbance units.
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Fig. 3.
NGF induces HO-1 expression.
A, heme oxygenase activity of PC12 cells treated with hemin,
NGF, actinomycin D (Act D), and cycloheximide
(CHX) as indicated. B, semiquantitative reverse
transcriptase-PCR showing induction of HO-1 mRNA. Upper
panel, HO-1 mRNA; lower panel, -actin mRNA
used for normalization. C, immunoblot showing induction of
HO-1 protein. Upper panel, blot with anti-HO-1 antibodies;
lower panel, blot with anti-protein-disulfide isomerase
(PDI) antibodies showing a similar amount of protein/lane.
For A-C, serum-starved cells were untreated or pretreated
with 50 µM cycloheximide or 10 µg/ml actinomycin D for
2 h prior to the addition of 50 µM hemin or 20 ng/ml
NGF for an additional 6 h. D, time course of HO-1
mRNA expression by NGF. Upper panel, semiquantitative
reverse transcriptase-PCR showing HO-1 mRNA; lower
panel,
-actin mRNA used for normalization. E,
time course of HO-1 protein expression by NGF. Upper panel,
immunoblot with anti-HO-1 antibodies; lower panel,
immunoblot with anti-protein-disulfide isomerase antibodies showing a
similar amount of protein/lane. F, densitometric
quantification of relative HO-1 mRNA and protein levels after
treatment with NGF. For D and E, serum-starved
cells were treated with 20 ng/ml NGF and then analyzed for HO-1
mRNA expression and protein at the indicated times.
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Fig. 4.
Pharmacological induction of HO-1 expression
with CoCl2 and moderate overexpression of HO-1 with a
retroviral expression vector attenuate 6-OHDA-induced oxidative
stress. A, dose effect of CoCl2 on HO-1
expression. Upper panel, immunoblot with anti-HO-1
antibodies; lower panel, immunoblot with
anti-protein-disulfide isomerase (PDI) antibodies showing a
similar amount of protein/lane. B, HE staining of
6-OHDA-treated cells in the presence of CoCl2 or bilirubin.
Serum-starved cells were pretreated with 100 µM
CoCl2 or 10 µM bilirubin for 12 h and
then treated with 40 µM 6-OHDA for 6 h and 2 µM HE for 1 h. Cells were analyzed for HE staining
by flow cytometry. Asterisks denote p < 0.001, control versus CoCl2- or bilirubin-treated groups.
C, immunoblot showing HO-1 levels in control and
LSN-HHO-1-infected cells. Densitometric analysis of three immunoblots
indicated a 2.3 ± 0.3-fold increase in HO-1 protein over the
level in control non-infected cells. D, HE staining of
control and HO-1-overexpressing cells treated with NGF and 6-OHDA.
Serum-starved cells were untreated or treated with 20 ng/ml NGF for
16 h and then treated with 40 µM 6-OHDA for 6 h. Cells were incubated with HE for 1 h and analyzed by flow
cytometry. a.u., absorbance units.
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Fig. 5.
Pharmacological inhibition of heme oxygenase
with tin-protoporphyrin and depletion of HO-1 with an antisense HO-1
retroviral construct abolish the protective effect of NGF against
6-OHDA-induced production of ROS. A, obstruction of the
antioxidant effect of NGF through inhibition of HO-1. Serum-starved
cells were co-treated with 20 ng/ml NGF and the indicated
concentrations of the HO-1 inhibitor tin-protoporphyrin
(SnPP) for 16 h. They were then treated with 40 µM 6-OHDA for 6 h as indicated. Finally, cells were
treated with 2 µM HE for 1 h and analyzed by flow
cytometry. B, basal and hemin-induced levels of HO-1 protein
in control PC12 cells and in two clones (AS1 and AS2) stably transduced
with antisense retroviral vector LSN-HOP-HHO-1-AS. Upper
panel, immunoblot with anti-HO-1 antibodies; lower
panel, immunoblot with anti-protein-disulfide isomerase
(PDI) antibodies showing a similar amount of protein/lane.
Serum-starved cells were untreated or treated with 50 µM
hemin for 6 h. Densitometric analysis of three immunoblots
indicated reductions in the levels of total HO-1 to 54 ± 8%
(AS1) and to 46 ± 12% (AS2) compared with control cells.
C, comparison of HE staining in control, AS1, and AS2 cells
treated with 6-OHDA in the presence or absence of NGF. Serum-starved
cells were untreated or pretreated with 20 ng/ml for 12 h and then
treated with 6-OHDA for 6 h. Cells were incubated with HE for
1 h and analyzed by flow cytometry. a.u., absorbance
units.
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Fig. 6.
NGF induces expression of HO-1 in a
PI3K-dependent manner. A, semiquantitative
reverse transcriptase-PCR of EGFP and myr-EGFP-Akt1 cells treated with
the PI3K inhibitor LY294002 and NGF as indicated. Upper
panel, HO-1 mRNA; lower panel,
glyceraldehyde-3-phosphate dehydrogenase (G3PDH) mRNA
used for normalization. B, immunoblots showing a partial
block of the NGF-induced increase in HO-1 protein levels in the
presence of LY294002. Upper panel, immunoblot with anti-HO-1
antibodies; lower panel, immunoblot with
anti-protein-disulfide isomerase (PDI) antibodies showing a
similar amount of protein/lane. For A and B,
serum-starved cells were pretreated with 40 µM LY294002
for 15 min and then stimulated with 20 ng/ml NGF for 6 and 12 h,
respectively. C, immunoblots showing HO-1 protein levels in
myr-EGFP-Akt1 cells and in untreated and 50 µM
hemin-treated EGFP cells. Upper panel, immunoblot with
anti-HO-1 antibodies; lower panel, immunoblot with
anti-protein-disulfide isomerase antibodies showing a similar amount of
protein/lane.
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Fig. 7.
The PI3K/Akt pathway mediates attenuation of
6-OHDA-generated ROS. A, the PI3K inhibitor LY294002
prevents attenuation of 6-OHDA-induced ROS by NGF. Serum-starved cells
were pretreated with 40 µM LY294002 (LY) for
15 min, stimulated with 20 ng/ml NGF for 12 h, and then submitted
to a 6-h induction with 6-OHDA and a 1-h incubation with 2 µM HE. HE staining was analyzed by flow cytometry of
10,000 cells/sample. B, cells overexpressing active Akt1
exhibit strong attenuation of 6-OHDA-induced production of ROS.
Serum-starved EGFP and myr-EGFP-Akt1 cells were treated with 40 µM 6-OHDA for 6 h and with 2 µM HE for
1 h. HE staining was analyzed by flow cytometry of 10,000 cells/sample. a.u., absorbance units.
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Fig. 8.
Overexpression of membrane-targeted active
Akt prevents apoptosis induced by 6-OHDA. A,
quantitative determination by flow cytometry of 6-OHDA-induced
apoptosis in cells stained with annexin V-PE and 7-AAD. EGFP and
myr-EGFP-Akt1 cells were treated with 40 µM 6-OHDA for
6 h and then incubated for 15 min with annexin V-PE (1:20 v/v) and
2 µM 7-AAD. A representative sample of 10,000 cells is
shown for each experimental condition. B, comparison of the
percentage of cells in apoptosis as determined from the lower
right quadrants in A.
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FOOTNOTES |
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* This work was supported in part by Grant SAF2001-0546 from the Spanish Ministry of Education and Grant 08.5/0048/2001 from the Comunidad Autónoma de Madrid.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.
§ Recipient of a fellowship (Formación de Personal Investigador) from the Spanish Ministry of Education.
Supported by Grant FEDER IF97/0602.
To whom correspondence should be addressed: Dept. de
Bioquímica, Facultad de Medicina, Universidad Autónoma de
Madrid, Arzobispo Morcillo 4, 28029 Madrid, Spain. Tel.:
34-91-397-5327; Fax: 34-91-585-4401; E-mail:
antonio.cuadrado@uam.es.
Published, JBC Papers in Press, February 10, 2003, DOI 10.1074/jbc.M209164200
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ABBREVIATIONS |
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The abbreviations used are: ROS, reactive oxygen species; NGF, nerve growth factor; PI3K, phosphatidylinositol 3-kinase; HO, heme oxygenase; 6-OHDA, 6-hydroxydopamine; EGFP, enhanced green fluorescent protein; myr, myristoylated; HE, hydroethidine; PE, phycoerythrin; 7-AAD, 7-aminoactinomycin D.
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