1 Instituto de Investigaciones Biomédicas "Alberto Sols",
Consejo Superior de Investigaciones Científicas-Universidad
Autónoma de Madrid (CSIC-UAM), Arturo Duperier 4, 28029 Madrid,
Spain
2 Centro de Investigaciones Biológicas, CSIC, Velázquez 144, 28006
Madrid, Spain
3 Departamento de Biología (Fisiología Animal), Universidad
Autónoma de Madrid, Carretera de Colmenar km. 15, Cantoblanco, 28049
Madrid, Spain
* Present address: Unidad de Investigación, Departamento de
Pediatría-UAM, Hospital Universitario Niño Jesús, Av.
Menéndez Pelayo 65, 28009 Madrid, Spain
Author for correspondence (e-mail:
yleon{at}iib.uam.es)
Accepted 14 October 2002
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Summary |
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Key words: p75 neurotrophin receptor (p75NTR), Ceramide, Caspase activation, Cell survival, Otic vesicle, Ceramide-1-phosphate
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Introduction |
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Inner ear ontogenesis is an attractive model system to analyse which
signals are implicated in the modulation of cell death and survival. The
vertebrate inner ear originates from the head ectoderm where the otic placode
is formed and invaginates to form the otic pit that later closes forming the
otic vesicle or otocyst. This is a transient embryonic structure that
undergoes a distinct period of intense cell proliferation [stages 18-22 in the
chicken (Hamburger and Hamilton,
1951)]. This proliferation precedes the differentiation of the
various cell types and compartments that will later form the adult inner ear
(Bissonnette and Fekete, 1996
).
In parallel, neuroblasts for the cochleo-vestibular ganglion (CVG) migrate out
from the medial wall of the otic vesicle. The CVG contains the afferent
neurons that connect the sensory epithelium of the inner ear to the central
nervous system (Hemond and Morest,
1991
). Concomitantly with these biological processes, in the otic
primordium and CVG there are areas of intense programmed cell death
(Alvarez and Navascués,
1990
; Sanz et al.,
1999a
). Complementary in vivo and in vitro studies point to
insulin-like growth factor-I (IGF-I) and NGF as two key diffusible factors
operating during inner ear development (reviewed by
Torres and Giraldez, 1998
;
Frago et al., 2000
).
NGF and the related neurotrophins are essential for cell survival and
maintenance of the nervous system during development
(Bibel and Barde, 2000;
de la Rosa and de Pablo,
2000
). The role of neurotrophins in neuronal survival is mainly
mediated by the activation of high-affinity Trk receptors
(Yoon et al., 1998
;
Klesse and Parada, 1999
).
However neurotrophins also bind the low-affinity p75 neurotrophin receptor
(p75NTR), which is a modulator of survival and death decisions
(Casaccia-Bonnefil et al.,
1999
; Barrett,
2000
; Lee et al.,
2001
). The p75NTR is structurally related to well known
death receptors, that is, the tumour necrosis factor-
(p55) and Fas
superfamily of receptors. Similarly, p75NTR possesses a death
domain that upon binding to ligand elicits the intracellular responses that
culminate in cell death (Beutler and van
Huffel, 1994
; Liepinsh et al.,
1997
). p75NTR-knockout mice present severe nervous
system defects (von Schack et al.,
2001
). In the past few years, the role of NGF as an inducer of
apoptosis has become evident. For example, treatment with NGF causes cell
death in cultures of mature oligodendrocytes
(Casaccia-Bonnefil et al.,
1996
) and in the developing chicken retina via an undefined
mechanism mediated by p75NTR (reviewed in
Frade and Barde, 1998
). NGF
also induces apoptotic cell death in organotypic cultures of otic vesicles
(Frago et al., 1998
).
Interestingly, NGF-induced cell death occurs in specific areas of the otic
vesicle and CVG, suggesting that during early inner ear organogenesis there is
strict control of NGF actions. The presence of p75NTR has been
reported at different stages of inner ear development in several animal
species, including chicken (Hallbook et
al., 1990
; von Bartheld et
al., 1991
; Schecterson and
Bothwell, 1994
; Wu and Oh,
1996
). The precise signalling pathway(s) used by p75NTR
to activate cell death remain unclear, but they may involve generation of
ceramide (Dobrowsky et al.,
1994
; Frago et al.,
1998
; Casaccia-Bonnefil et
al., 1996
; Lievremont et al.,
1999
) and activation of caspases-1, -2 and -3
(Gu et al., 1999
) as well as
cyclin-dependent kinases (Frade,
2000
).
IGF-I is a pleiotropic factor for the epithelial and neuronal cells of the
inner ear, which is where IGF-I and its type 1 receptor are expressed
throughout development (León et
al., 1999). The importance of IGF-I in ear development is stressed
by the fact that deficiency in IGF-I results in sensorineural deafness in
humans (Woods et al., 1996
;
Woods et al., 1997
). Likewise,
the lack of IGF-I in mice severely affects postnatal survival, differentiation
and maturation of the cochlear ganglion cells and causes abnormal innervation
of the sensory cells in the organ of Corti
(Camarero et al., 2001
).
Furthermore, IGF-I is a survival and growth factor capable of blocking
apoptosis in organotypic cultures of chick otic vesicles
(Frago et al., 1998
). By
binding to its high-affinity tyrosine kinase receptor, IGF-I activates the
Raf/MAPK cascade and induces the expression of Fos, Jun and proliferating cell
nuclear antigen (PCNA), which leads to cell growth
(Frago et al., 2000
;
Sanz et al., 1999b
). However,
little is known about the intracellular pathways elicited by IGF-I to promote
cell survival during development. One of the pathways by which IGF-I exerts
its anti-apoptotic effect in different cell types including cells of the
nervous system is the activation of the Akt/Protein kinase B pathway
(Dudek et al., 1997
). Akt
family members have been shown to be required for normal development in flies
and Caenorhabditis elegans
(Paradis and Ruvkun, 1998
;
Verdu et al., 1999
), and
Akt1-null mice present defects in both foetal and postnatal growth
(Cho et al., 2001
).
Here we have explored the intracellular signalling mechanisms elicited by NGF to induce apoptosis in the developing inner ear and how they are balanced by survival signals activated by IGF-I. We show that (i) p75NTR mediates NGF pro-apoptotic effects, including ceramide generation, caspase activation and DNA fragmentation; (ii) NGF induces ceramide generation via synthesis de novo and acid sphingomyelinase activation; (iii) NGF induces the activation of initiator and effector caspases and the hydrolysis of poly-(ADP ribose) polymerase (PARP); (iv) only initiator caspases are involved in ceramide generation; and (v) IGF-I prevents NGF actions, at least in part, by decreasing intracellular ceramide levels and activating Akt.
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Materials and Methods |
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The caspase inhibitors, zVAD-fmk (zVAD) (Calbiochem) and z-DEVD-fmk (DEVD) (Enzyme Systems Products, Livermore, CA), contain a peptide recognition sequence attached to a functional fluoromethylketone (fmk) group. These inhibitors are cell permeable and act by binding irreversibly to the active site of caspases. zVAD is a pan-caspase inhibitor, and DEVD is an inhibitor of ICE/CED-3 family of cysteine proteases.
Preparation of organotypic cultures
Fertilized eggs from White-Leghorn hens, purchased at a local farm, were
incubated at 38°C in a humidified atmosphere and were staged according to
Hamburger and Hamilton (HH) criteria
(Hamburger and Hamilton,
1951). Otic vesicles were dissected from chick embryos
corresponding to HH18 (embryonic day 2.5) and cultured in serum-free M199
medium with Earle's salts (Biowhitaker, Walkersville, MD) supplemented with 2
mM glutamine (Biowhitaker) and antibiotics [50 IU/ml penicillin (Ern,
Barcelona, Spain) and 50 µg/ml streptomycin (CEPA, Madrid, Spain)].
Treatments with 4 nM NGF, 1 nM IGF-I, 5 µM C2-Cer, 5 µM
dihydroceramide or 25 µM Cer-1-P were performed in the absence or presence
of the different antibodies or inhibitors at 37°C in a water-saturated
atmosphere containing 5% CO. C2-Cer and manumycin A were prepared
in DMSO and desipramine was prepared in methanol. The final concentration of
DMSO or methanol in culture medium was 0.01%, which had no detectable effect
on otic vesicle cultures. Otic vesicles cultured in medium without additions
or, when indicated, with solvents, were taken as control values.
Western blot analysis
For western blotting, HH18 otic vesicles (1, 8 or 12 otic
vesicles/datapoint for PCNA, PARP or Akt immunodetection, respectively) were
made quiescent by incubation overnight in serum-free medium. Treatments were
carried out at 37°C for 16 hours for PCNA and PARP analysis and 30 minutes
for the Akt study. Afterwards, otic vesicles were homogenised in Laemmli
Buffer (1.5x) with 1 mM phenylmethylsulfonyl-fluoride and frozen
immediately. Gels were loaded with solutions containing equal amounts of
proteins. Otic vesicle proteins were subjected to SDS-PAGE on 8%
polyacrylamide gels for PARP or 12% gels for PCNA and Akt analysis. The
electrophoresed proteins were transferred onto PVDF membranes using a Bio-Rad
Trans Blot according to the manufacturer's instructions. After incubation with
blocking solution (5% non-fat dry milk in Tris-buffered saline with 0.1% Tween
20), blots were probed with the appropriate specific primary antibodies for 2
hours at room temperature or overnight at 4°C. All antibodies were diluted
in blocking solution except anti-phospho-Akt antibodies, which were diluted in
Tris-buffered saline with 0.1% Tween 20 containing 5% bovine serum albumine.
Blots were subsequently washed and then incubated with the adequate secondary
antibodies conjugated with peroxidase for 1 hour at room temperature. The
immunoblots were developed with a chemiluminiscence system (Dupont-NEN,
Boston, MA) and exposed to X-ray film (Konica). Films were scanned in a Studio
Scan II (Agfa) and the bands were quantified by densitometry with NIH Image
1.59 software.
Explant labelling and determination of ceramide levels
Otic vesicles were isolated from HH18 chick embryos and labelled with 25
µCi/ml of [3H]palmitic acid for 24 hours in the presence of 1%
fetal bovine serum. They were then washed with PBS, placed in serum-free
medium and stimulated at 37°C with 4 nM NGF for 3 hours. Cellular lipids
were extracted, and labelled ceramide was purified as reported
(Frago et al., 1998). Briefly,
lipids were extracted according to Bligh and Dyer
(Bligh and Dyer, 1959
), except
that phases were separated by adding 2 M KCl in 0.2 M
H3PO4 instead of water. The organic phase was dried
under a N2 stream and the lipid extracts were applied to thin-layer
plates (silica Gel G60 t.l.c.), which were developed twice with
chloroform:methanol:acetic acid (9:1:1, by volume). The material that
co-migrated with cold ceramide standards was scraped and extracted with 1 ml
methanol/H2O (1:1 by volume). Radioactivity associated with the
spots was determined by scintillation counting.
Assessment of apoptosis
Two techniques were used to study apoptotic cell death: TUNEL and DNA
fragment ELISA. Distribution of apoptotic cells in the otic vesicle was
determined by Tdt-mediated dUTP nick end labelling (TUNEL) of the fragmented
DNA with the modifications reported previously
(Blaschke et al., 1996) and
adapted to whole organ labelling (Frago et
al., 1998
; Díaz et al.,
1999
). Briefly, otic vesicles were fixed overnight with 4% (w/v)
paraformaldehyde in 0.1 M PBS pH 7.4 and permeated by four incubations with 1%
(w/v) Triton X-100 in PBS for 30 minutes at room temperature and one
incubation with 20 µg/ml proteinase K (Roche Molecular Biochemicals) for 10
minutes at 37°C. The otic vesicles were then incubated with the terminal
deoxynucleotidyl-transferase buffer for 30 minutes at 37°C and
subsequently incubated with 0.1 U/µl terminal deoxynucleotidyl-transferase
and 10 µM biotin-16-deoxy-UTP (Roche Molecular Biochemicals) for 2.5 hours
at 37°C. The reaction was stopped by incubation with 2 mM EDTA in PBS for
1 hour at 65°C. TUNEL labelling was visualized by incubation with
Cy2-streptavidin (Amersham Pharmacia Biotech, Rainham, Essex, UK), which was
followed by transparentation with 70% glycerol in PBS (v/v) and mounting for
epifluorescence. Fluorescence was analysed in a MRC1024 BioRad confocal
microscope. At least five otic vesicles per condition were assayed in three
different experiments. In parallel, TUNEL assays on 25 µm cryostat sections
of HH18 chicken embryos were performed essentially by following manufacturer's
instructions (In situ Cell Death detection kit, POD, Roche Molecular
Biochemicals), as described previously
(Sanz et al., 1999a
). Briefly,
sections were fixed in 4% paraformaldehyde (w/v) for 20 minutes and then
rinsed in 0.1% Triton X-100 with 0.1% sodium citrate for 2 minutes. Sections
were then incubated with the terminal deoxynucleotidyl transferase enzyme in
buffer containing fluorescein nucleotides for 1 hour at 37°C. The TUNEL
signal was visualized with an antibody antifluorescein coupled to peroxidase
for 30 minutes at 37°C. At least nine embryos were sampled in three
different assays.
Otocyst cell death was quantified by using the Cell Death Detection ELISA (Roche Molecular Biochemicals), which was based on the detection of histone-associated DNA fragment in the cytoplasm of cells. Quiescent HH18 otic vesicles were cultured in serum-free medium with different additives for 16 hours and then processed for apoptosis determination. A single HH18 cultured otic vesicle was homogenised in 100 µl of the supplied incubation buffer and the cell extracts subjected to ELISA determination following basically the instructions of the supplier.
Whole mount in situ hybridization
In situ hybridisation was performed on chicken embryos as described
previously (Wilkinson, 1992).
A single-stranded chicken p75NTR RNA probe (A.
Rodríguez-Tebar, Instituto Cajal, Madrid, Spain) was prepared by
transcription of the linearized plasmid pKS-p75 with XbaI, using T3
RNA polymerase (Promega, Madison, WI)
(Large et al., 1989
). The
control sense probe was prepared by using T7 RNA polymerase (Promega, Madison,
WI) after linearizing pKS-p75 with HindIII. Embryos were incubated
overnight at 70°C with 1 µg/ml digoxigenin-labelled RNA
p75NTR probe in hybridization mix (50% formamide, 1.3xSSC pH
4.5, 5 mM EDTA, 50 µg/ml yeast RNA, 0.2% Tween 20, 0.5% CHAPS and 100
µg/ml heparin). To elicit colour, embryos were incubated with a 1/2000
dilution of antidigoxigenin-alkaline phosphatase antibody (Roche Molecular
Biochemicals). Colour was developed with 0.26 mg/ml nitroblue tetrazolium
chloride and 0.175 mg/ml 5-bromo-4-chloro-3 indolyl-phosphate prepared in NTMT
(100 mM NaCl, 100 mM Tris-HCl pH 9.5, 50 mM MgCl2 and 0.1% Tween
20). For histological examination, embryos were post-fixed in 4%
paraformaldehyde, embedded in gelatine-albumin and sectioned with a vibratome
(Leica VT 1000M, Heidelberg, Germany) at a thickness of 60 µm. At least 12
embryos were sampled in three different experiments.
Immunocytochemistry
Immunocytochemistry was performed on frozen sections obtained from HH18
chicken embryos fixed overnight in 4% paraformaldehyde (w/v), dehydrated in
30% sucrose and then included in Tissue Teck (Miles Diagnostics Div.,
Kankakee, IL). Frozen embryos were sectioned at a thickness of 20 µm with a
cryostat (Leitz, Jena, Germany). Sections were placed on polylysine-coated
slides and processed for p75NTR receptor analysis following the
procedures previously described
(Casaccia-Bonnefil et al.,
1996; Sanz et al.,
1999a
). Briefly, sections were incubated in 3%
H2O2 in methanol for 20 minutes and then blocked in a
solution of 0.1% Triton X-100 in PBS containing 10% goat serum for 1 hour.
Sections were left incubating overnight with rabbit polyclonal
anti-p75NTR antibody (1/1000 in the blocking solution) in a humid
chamber at 4°C. Afterwards sections were incubated in EnVision+
anti-rabbit peroxidase-conjugated secondary antibodies (Dako, Copenhagen,
Denmark) for 30 minutes and peroxidase was developed with 0.5 mg/ml
diaminobenzidine tetrahydrochloride (Sigma) and 0.01%
H2O2. At least 10 embryos were analyzed in three
different experiments.
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Results |
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The first step was to study the resulting effect when both factors were acting together on cultured otic vesicles. As shown in Fig. 1, NGF increased the number of TUNEL-positive cells, particularly at the ventromedial wall of the otic vesicle, where the CVG is emerging (Fig. 1, compare B with A) and within the CVG. C2-Cer addition induced cell death with a less restricted pattern (Fig. 1C). IGF-I not only effectively reduced cell death caused by serum deprivation (Fig. 1, compare D with A), but it was also able to prevent apoptosis induced by NGF and C2-Cer (Fig. 1E,F).
|
Expression of p75NTR in the developing inner ear
Once the epistatic effect of IGF-I on NGF was demonstrated, we
characterized some of the pathways activated by NGF during cell death
induction in the otic vesicles to later analyze the sites blocked by IGF-I.
The presence of NGF receptors in the otic vesicle epithelium and CVG has been
reported in several species (Hallbook et
al., 1990; von Bartheld et
al., 1991
; Schecterson and
Bothwell, 1994
; Wu and Oh,
1996
). We extended these reports by assessing the presence of
p75NTR transcripts in HH18 chicken otic vesicles by whole mount in
situ hybridization and immunohistochemistry
(Fig. 2). The expression of
p75NTR transcripts and protein was high in restricted areas of the
otic vesicle epithelium and within the CVG
(Fig. 2A,B). p75NTR
expression was also high in the mesenchyme surrounding the otocyst.
Furthermore, apoptotic cell death was detected in the same areas both in vivo
and in cultured otic vesicles treated with NGF
(Fig. 2C-E). Therefore,
p75NTR was expressed within the areas of high cell death in the
otocyst at the developmental stage studied.
|
p75NTR and caspase activation mediate NGF-induced
apoptosis in the otic vesicle
The involvement of p75NTR in NGF pro-apoptotic actions was
studied by using the 9651 anti-p75NTR antibody, which effectively
blocks NGF binding to p75NTR
(Huber and Chao, 1995). The
levels of cytoplasm-soluble nucleosomes were measured in otic vesicle extracts
to quantify cell death caused by treatment with NGF or NGF plus
anti-p75NTR antibodies. Table
1 shows that apoptosis induced by NGF was completely prevented by
the 9651 anti-p75NTR antibody but not by treatment with the control
non-blocking 9992 anti-p75NTR antibody. These results indicate that
in the otic vesicle the apoptotic response to NGF requires binding to
p75NTR. In addition, the specificity of NGF actions was confirmed
by culturing the otic vesicle explants in the presence of NGF plus anti-NGF
antibody. Under these conditions, NGF did not induce cell death. Treatment of
otic vesicles with any of these antibodies alone had no appreciable effect
(Table 1). Therefore, NGF
effects on otic vesicle apoptosis are specific for this neurotrophic factor
and are mediated through binding to p75NTR.
|
To evaluate the implication of caspases in p75NTR-dependent NGF-induced cell death, otic vesicles were cultured in the presence of caspase inhibitors, which completely suppressed death induced by NGF (Table 2). Treatment of otic vesicles with either zVAD (25 µM) or DEVD (75 µM) completely blocked NGF-induced apoptosis, whereas the addition of zVAD or DEVD had no effect on basal cell death. Caspase inhibitors also prevented C2-Cer-induced cell death. The specificity of C2-Cer actions was tested using dihydroceramide, an inactive synthetic analogue of C2-Cer. The addition of 5 µM dihydroceramide had no effect on the relative amount of oligonucleosomes in the cytoplasm, indicating that C2-Cer actions were specific (Table 2).
|
TUNEL staining of cultured otic vesicles confirmed these results (Fig. 3). Otic vesicles cultured in serum-free medium presented basal levels of cell death, whereas NGF treatment increased the number of TUNEL-positive cells (Fig. 3A,B). By contrast, pre-treatment with either anti-NGF antibodies or anti-p75NTR blocking antibodies led to a significant reduction in cell death when compared with otic vesicles cultured with NGF alone (Fig. 3B-D). Pre-treatment of the otic vesicles with caspase inhibitors also blocked NGF-induced apoptosis in the otic vesicle epithelium and the CVG (Fig. 3F,H). Treatment with either the antibodies or the inhibitors alone had no effect (data not shown and Fig. 3E,G, respectively).
|
To further explore NGF-p75NTR downstream signalling,
intracellular ceramide levels and PARP cleavage were determined in the absence
or presence of anti-p75NTR antibodies, anti-NGF antibodies or
caspase inhibitors (Fig. 4).
Treatment with either anti-NGF or 9651 anti-p75NTR antibodies
impaired NGF induction of ceramide accumulation
(Fig. 4A). The next step was to
study the roles of the caspase subfamilies in the generation of endogenous
ceramide. Fig. 4A shows that
zVAD, but not DEVD, was able to impair the increase in ceramide levels caused
by NGF. Therefore, these data suggest that the increase in the intracellular
ceramide content produced by NGF is secondary to the activation of a subset of
zVAD-sensitive DEVD-insensitive caspases, most probably initiator caspases.
The degree of PARP cleavage to the 85 kDa PARP fragment, a marker of
caspase-3-dependent apoptosis (Lazebnik et
al., 1994), was also determined in otic vesicles cultured under
different conditions. NGF increased the proteolytic cleavage of PARP by
2.6-fold, which was prevented by pre-treatment with either blocking
anti-p75NTR antibodies or caspase inhibitors
(Fig. 4B). PARP cleavage was
also increased in otic vesicles cultured in the presence of C2-Cer
by 4.5-fold, and this was again prevented by treatment with caspase inhibitors
(Fig. 4C). Taken together these
results suggest that following NGF binding to p75NTR, initiator
caspases are activated. This activation will lead to an increase in
intracellular ceramide content that in turn will stimulate effector
caspase-3-like proteases, leading to PARP degradation, DNA fragmentation and
cell death.
|
NGF-induced cell death is dependent on ceramide generation
Next we examined the intracellular source of the ceramide released by the
action of NGF (Fig. 4A). FB1
(50 µM), an inhibitor of de novo ceramide synthesis, reduced the
NGF-dependent increase in ceramide levels by 50% and was also able to impair
NGF-dependent cell death (Table
3). Desipramine (10 µM), an acid sphingomyelinase inhibitor,
was also able to impair NGF actions by decreasing ceramide levels by 52% and
blocking cell death (Table 3).
On the other hand, the neutral sphingomyelinase inhibitor manumycin A did not
cause any effect at the doses tested (10 to 100 µM, data not shown).
|
IGF-I activates Akt and blocks NGF-induced ceramide increase
Finally, we explored the crosstalk between the IGF-I and NGF intracellular
pathways. Treatment of cultured otic vesicles with IGF-I prevented NGF-induced
ceramide accumulation (Fig. 4A; Fig. 5A). Ceramide is
phosphorylated in vivo by a ceramide kinase to give Cer-1-P
(Dressler and Kolesnick,
1990), which is a cytoprotector for the otocyst
(Frago et al., 1998
). In
vitro, bacterial DGK carries out the same reaction
(Schneider and Kennedy, 1973
).
Ceramide kinase has been recently cloned, purified and its biochemical
activity characterized in vitro (Sugiura
et al., 2002
). Ceramide kinase has a DGK catalytic domain
(Sugiura et al., 2002
), which
is the target of the DGK inhibitor R59949
(Jiang et al., 2000
). To test
whether IGF-I is further controlling intracellular ceramide levels by
modulating ceramide phosphorylation, otic vesicle cultures were treated with
R59949, which produced a slight increase in basal apoptotic cell death but was
unable to affect IGF-I actions (Table
4). The cytoprotective effect of Cer-1-P is shown for comparison
(Table 4). In the presence of
either NGF or C2-Cer, R59949 effectively impaired the survival
actions of IGF-I. Moreover, treatment with R59949 blocked the actions of IGF-I
on NGF-induced increase in ceramide levels
(Fig. 5A) and on the activation
of JNK (data not shown). IGF-I prevents JNK activation induced by both serum
deprivation and NGF (Sanz et al.,
1999a
). These results suggest that the protective effect of IGF-I
on cell death may be mediated by inducing ceramide phosphorylation.
|
|
Activation of the pathway initiated by phosphorylation of the Akt
serine/threonine kinase by IGF-I is central to cell survival
(Dudek et al., 1997;
Zheng et al., 2000
). In
cultured otic vesicles, IGF-I effectively stimulated the phosphorylation of
Akt at Ser473 (4.7-fold) and Thr308 (4-fold) residues
(Fig. 5B and data not shown,
respectively). NGF and C2-Cer did not induce changes in the levels
of Akt phosphorylation and were unable to alter the induction by IGF-I of the
phosphorylation at Ser473-Akt (4.1- and 3.5-fold, respectively) and Thr308
(3.8- and 2.3-fold, respectively) (Fig.
5B and data not shown). However, pre-treatment with R59949 plus
NGF or C2-Cer decreased IGF-I-induced Ser473- and Thr308-Akt
phosphorylation (2.8- and 2.2-fold, respectively for Ser473-Akt
phosphorylation; 2.2- and 1.2-fold for Thr308-Akt phosphorylation), whereas in
the absence of IGF-I R59949 alone or in combination with NGF had no effect
(data not shown). Treatment with Cer-1-P also increased Akt phosphorylation
levels by 2.7- and 1.9- fold at Ser473 and Thr308 residues, respectively. On
the other hand, phosphatidic acid (10-50 µM), the natural product of DGK,
had no effect on Akt phosphorylation (25 µM, 0.9±0.2 fold) at the
same time points. Changes in the degree of phosphorylation of Akt were
paralleled by changes in the levels of PCNA, a marker of cell proliferation
(Fig. 5B).
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Discussion |
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Cell death, which normally takes the route of apoptosis, is a physiological
process during development and morphogenesis
(Raff et al., 1993). Apoptosis
can be induced or prevented by a variety of stimuli that activate different
intracellular signalling pathways, which converge to activate or inhibit a
common pool of executioner molecules. Analysis of many linear intracellular
signal transduction and apoptotic pathways has been the object of intense
research. But how and where they intersect and whether this crosstalk results
in synergy or antagonism is not known in most cellular contexts. Therefore,
determining the sequence of events and the interdependencies involved in
apoptosis signalling is an area of active research.
Among the neuronal pro-apoptotic factors, considerable attention is
presently focused on NGF and its low-affinity receptor, p75NTR. It
has been proposed that the relative levels of high- and low-affinity NGF
receptors determine cell fate (Lee et al.,
2001; Chao and Bothwell,
2002
). The cellular response to NGF will, therefore, depend on the
developmental modulation of NGF receptors expression. In the present study, we
show that in the otic vesicle NGF-induced apoptosis is mediated by binding to
p75NTR. A similar situation has been reported for embryonic retinal
cells and postnatal oligodendrocytes where activation of p75NTR
increases cellular apoptosis
(Casaccia-Bonnefil et al.,
1996
; Frade et al.,
1996
; Frade, 2000
;
González-Hoyuela et al.,
2001
). In the otocyst, after binding to p75NTR, NGF
increases ceramide levels, and this increase can be reduced by treatment with
inhibitors of acid sphingomyelinase and de novo synthesis.
p75NTR-induced apoptosis has been shown to trigger ceramide
generation in different neural cell types including oligodendrocytes and
neuroblastoma cells (Dobrowsky et al.,
1994
; Dobrowsky et al.,
1995
; Casaccia-Bonnefil et
al., 1996
; Lievremont et al.,
1999
). Although earlier studies pointed to a membrane-neutral
sphingomyelinase associated with p75NTR as the enzyme responsible
for ceramide increase (Brann et al.,
1999
; Dobrowsky and Carter,
2000
), such an enzyme has not yet been characterized and our
results indicate that, in the otic vesicle, NGF actions are dependent on both
the full activity of an acid sphingomyelinase and on an increase in de novo
ceramide synthesis. These results are in agreement with the sustained increase
in ceramide levels (up to 4 hours) induced by NGF in this system
(Frago et al., 1998
). Acid
sphingomyelinase participates in stress-activated and developmental apoptosis
(Peña et al., 1997
;
Morita et al., 2000
;
Cutler and Mattson, 2001
) and
p75NTR overexpression induces cell survival in human Niemann-Pick
fibroblasts, which lack acid sphingomyelinase activity
(Roux et al., 2001
). On the
other hand, de-novo-synthesized ceramide has been reported to be crucial for
apoptosis in different cellular settings including during early neural
differentiation (Herget et al.,
2000
; Gomez del Pulgar et al.,
2002
).
Several studies indicate that ceramide generation is essential for
apoptosis, but there are discrepancies concerning whether ceramide generation
is upstream or downstream of caspases activation
(Chinnaiyan et al., 1996;
Mizushima et al., 1996
;
Hartfield et al., 1998
). Some
groups studying secondary cultures of adult cells have concluded that ceramide
action is downstream of the initiator caspases but upstream from the
executioner caspases, such as caspase-3
(Sweeney et al., 1998
;
Grullich et al., 2000
;
Craighead et al., 2000
).
Caspase-3 is thought to be primarily required for the nuclear changes that
occur during apoptosis (Oppenheim et al.,
2001
). Furthermore, recent studies show that ceramide induces
non-apoptotic programmed cell death with necrotic-like morphology
(Mochizuki et al., 2002
). We
have studied NGF-induced ceramide generation in the presence of caspase
inhibitors in organotypic cultures of the developing otocyst. We show that
after NGF binding to p75NTR only zVAD was capable of blocking
NGF-induced ceramide release. On the other hand, PARP-cleavage and cell death
were impaired by both zVAD and DEVD, suggesting that the activation of both
initiator and caspase-3-like proteases is required for PARP hydrolysis and
cell death. Therefore, the activation of the caspase-3 occurs downstream of
the increase in intracellular ceramide content produced in response to NGF.
These data suggest that an increase in ceramide could be a secondary signal
activated by caspases and capable of further activating these proteases. The
ceramide increase may, therefore, be a reinforcement loop to commit cells to
apoptosis.
In the otic vesicle, the survival actions of IGF-I are independent from the
stimuli responsible for the initiation of apoptosis, protecting it from serum
withdrawal, NGF and C2-Cer-induced cell death.
Igf-1-knockout mice show increased apoptosis and activated caspase-3
in the cochlear ganglia (Camarero et al.,
2001). By contrast, transgenic mice that overexpress IGF-I present
decreased levels of activated caspase-3 and a general impairment in apoptotic
pathways (Chrysis et al.,
2001
). In parallel to these studies, it is known that a major
pathway for the anti-apoptotic actions of IGF-I is the activation of the Akt
kinase (Dudek et al., 1997
).
Phosphorylation of Akt at threonine 308 by phosphoinositide-dependent kinase 1
is followed by autophosphorylation at serine 473 to produce an activated Akt
whose role is to facilitate survival by phosphorylation of downstream
substrates (Brazil and Hemmings,
2001
). Akt kinase plays a crucial role in supporting NGF-dependent
survival through Trk receptors, whereas activation of the p75NTR
receptor can initiate a cell death cascade in neurons and glial cells
(Kaplan and Miller, 2000
).
Here we show that IGF-I acts at different levels to protect otic vesicles from
NGF-induced cell death. First, IGF-I blocks the intracellular increase in
ceramide generation elicited by NGF and second, IGF-I activates Akt.
Therefore, the protective role of IGF-I in p75NTR-dependent cell
death is, at least in part, mediated by decreasing ceramide levels with a
concomitant activation of Akt phosphorylation. Cer-1-P, a molecule that is
reported to be a cytoprotector for otic vesicle explants, also activated Akt.
The crosstalk between PI 3-kinase, an enzyme upstream in the intracellular
cascade of Akt, and the sphingomyelinase pathways has been shown to be
important for the balance between cell survival and death decisions
(Burow et al., 2000
). Indeed,
PI 3-kinase downregulation by stress is dependent on acid sphingomyelinase
(Zundel and Giaccia, 1998
).
C2-Cer has been reported to inhibit Akt activity
(Schubert et al., 2000
;
Teruel et al., 2001
); however,
in the otic vesicle, IGF-I is able to protect the otocyst from
C2-Cer-induced apoptosis, suggesting that in addition to Akt
activation there is another mechanism(s) by which IGF-I blocks cell death
induced by exogenous C2-Cer. One possible fate for ceramide is
conversion to Cer-1-P by ceramide kinase, which has a DGK catalytic domain
(Sugiura et al., 2002
). By
using R59949 we have explored the possibility that the protective effects of
IGF-I on otic vesicle survival could be due to an increase in Cer-1-P. In the
presence of R59949, the protective action of IGF-I against apoptosis induced
by NGF or C2-Cer was lost, and this is associated with an increase
in NGF-induced endogenous ceramide levels. Other lipid mediators such as
phosphatidic acid or sphingosine-1-phosphate have no significant survival
actions in this system as shown here and previously
(Frago et al., 1998
). These
data suggest that IGF-I enhances the conversion of the pro-apoptotic ceramide
to its phosphorylated cytoprotective metabolite Cer-1-P; therefore, the
relative levels of ceramide and Cer-1-P could be an intracellular indicator
for cell death/survival decisions.
Our results indicate that during the early development of the chicken inner ear the signalling pathways induced by NGF and IGF-I form a complex network that regulates apoptotic cell death, a likely scenario for the regulation of early neural cell death (Fig. 6). We provide evidence that the molecular basis for the restricted apoptotic response to NGF in the otocyst involves modulation of the activation of death p75NTR versus survival receptors. Activation of survival signalling pathways implies blockage of the apoptotic pathways at several levels. Hence, some aspects of the regulation of inner ear development appear to occur thanks to the crosstalk between NGF and IGF-I signalling. We propose that the dynamic balance between levels of ceramide metabolites and the consequent regulation of Akt phosphorylation are important factors that determine whether a cell survives or dies.
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