The C-terminal Fragment of Presenilin 2 Triggers p53-mediated
Staurosporine-induced Apoptosis, a Function Independent of the
Presenilinase-derived N-terminal Counterpart*
Cristine Alves
da Costa
§,
Mark P.
Mattson¶,
Karine
Ancolio
, and
Frédéric
Checler§
From the
Intitut de Pharmacologie Moléculaire
et Cellulaire of CNRS, UMR6097, Valbonne 06560, France and
¶ NIA Gerontology Research Center, National Institutes of Health,
Baltimore, Maryland 21224
Received for publication, December 5, 2002, and in revised form, January 24, 2003
 |
ABSTRACT |
Mutations on presenilins are responsible for most
of familial forms of Alzheimer's disease. These holoproteins undergo
rapid maturation by presenilinase mainly in the endoplasmic reticulum, leading to the production of N- and C-terminal fragments. We show first
that overexpression of the presenilinase-derived maturation product of
presenilin 2 (CTF-PS2) increases A
recovery, the production of which
is almost abolished by a caspase 3 inhibitor and increased by
staurosporine. This and the observation that the apoptotic inducer
staurosporine enhances CTF-PS2 degradation clearly
link CTF-PS2 to apoptotic cascade effectors. This prompted us to
analyze the putative ability of CTF-PS2 to modulate cell death. CTF-PS2 overexpression decreases cell viability and augments both caspase 3 activity and immunoreactivity. This is accompanied by lowered bcl2-like immunoreactivity and increased
poly(ADP-ribose) polymerase cleavage and cytochrome
c translocation into the cytosol. Interestingly, CTF-PS2-induced caspase 3 activation is prevented by pifithrin-
, a
selective blocker of p53 transcriptional activity. On line with the
latter data, CTF-PS2 drastically increases p53 immunoreactivity and
transcriptional activity. Of most interest is our observation that
CTF-PS2 expression also triggers increased caspase 3 activity and
immunoreactivity in fibroblasts in which presenilins had been deleted.
Therefore, CTF-PS2 could modulate cell death out of the NTF/CTF
heterodimeric complex thought to correspond to the biologically functional entity. This is the first direct demonstration that CTF-PS2
could exhibit some of its functions in the absence of the presenilin 2 N-terminal fragment (NTF-PS2) counterpart derived from the
presenilinase cleavage.
 |
INTRODUCTION |
Presenilins 1 and 2 (PS1 and
PS2)1 have been the center of
a huge amount of studies because most of the familial forms of
Alzheimer's disease are due to inherited mutations on genes coding for
these two parent proteins (for reviews, see Refs. 1-4). PS1 and PS2 are highly homologous transmembrane proteins mainly located in the
early compartments of the secretory pathway where they undergo various
post-transcriptional modifications (5). Particularly interesting is the
susceptibility of PS to an endoproteolytic cleavage, giving rise to
N-terminal and C-terminal (CTF) fragments (6-9) by a yet unknown
protease referred to as presenilinase. This proteolytic event appears
of major importance because intact PS holoproteins are poorly
detectable in the brain of affected patients. The two proteolytic
counterparts appear to accumulate with a 1/1 stoichiometry (7), and
this complex is thought to exhibit the PS-mediated phenotypes.
Among other biological functions, PSs have been shown to control the
production of the amyloid
-peptides and to modulate cell death in
response to various apoptotic stimuli (10, 11). Interestingly, these
two functions are altered by Alzheimer's disease-related pathogenic
mutations. Thus, all mutations increase the production of amyloid
-peptides (12) and, more specifically, the 42-amino-acid-long A
that appears to be more implicated in the pathology than its shorter
counterpart of 40 residues. More recently, we showed that wild type PS2
and, more potently, mutated PS2, trigger caspase 3 activation by a
p53-dependent mechanism (13). This was accompanied by a
decrease in PS1 expression, in agreement with a previous report showing
that p53 depletion increases PS1 expression and delayed apoptotic cell
death (14).
Interestingly, the presenilinase-derived CTF fragment undergoes
phosphorylation events (15, 16) that appear to affect its
susceptibility to caspase-mediated proteolysis (17-19). This could
suggest that at least part of the PS-associated modulation of cell
death could be related to the production of the CTF or its
caspase-derived fragment referred to as CTFcas. Therefore, two
main questions at least remained to be addressed. First, what are the
mechanisms by which the CTF fragment could modulate the apoptotic cell
death? Second, is CTF acting through the interaction of its N-terminal
counterpart, or is it able to trigger a phenotype in absence of
N-terminal fragments?
Here we establish that the overexpression of CTF-PS2 augments A
, the
production of which is prevented by caspase 3 inhibitors. Furthermore,
CTF-PS2 increases caspase 3 expression and activity and elevates both
expression and transcriptional activity of p53. Of most interest, we
show that this CTF-PS2-related phenotype was still observed in
PS-deficient cells, indicating that CTF-PS2 could be biologically
active per se in the absence of any heterodimeric interaction with its N-terminal counterpart.
 |
MATERIALS AND METHODS |
Cells Systems--
HEK293 cells stably expressing the C-terminal
fragment of PS2 (CTF-PS2) were obtained and cultured as previously
described (20). Mice in which the genes of PS1 and PS2 were deleted
(kindly provided by Drs. Bart De Strooper (Center for Human Genetics, Leuven, Belgium) and Paul Saftig (Christian Albrecht University, Kiel,
Germany)) have been previously described (21). Mice fibroblasts were obtained and immortalized with SV40 large T antigen according to
the procedure previously reported (22) and cultured as previously reported (23). Transient transfection of fibroblasts with the cDNA
encoding either for CTF-PS2 or empty vector was performed with DAC30
according to manufacturer's recommendations (Eurogentec, France).
CTF-PS2 expression in stably and transiently transfected cells was
checked by Western blot by means of the Ab333 antibodies (rabbit
polyclonal anti-C-terminal fragment of PS2 (provided by W. Araki and T. Tabira) as previously described (24).
Flow Cytometry Analysis of Propidium Iodide
Incorporation--
HEK293 cells were grown in 6-well plates and
incubated for 24 h at 37 °C in the presence or absence of 2 µM staurosporine. Cells were harvested, pelleted by
centrifugation at 1000 × g for 10 min at 4 °C,
gently resuspended in 500 µl of 0.1% sodium citrate buffer
containing 50 µg/ml propidium iodide, and incubated overnight under
agitation. The propidium iodide fluorescence of individual nuclei was
measured using a FACScan flow cytometer (program CellQuest, BD
Biosciences) as described (13).
Terminal dUTP Nick-end Labeling (TUNEL) Analysis--
Cells were
cultured in 6-well plates and then treated or not with 2 µM staurosporine for 24 h. After treatment cells
were fixed for 30 min with 4% paraformaldehyde, rinsed in PBS,
permeabilized overnight with 70% ethanol, and then processed for the
dUTP nick-end labeling TUNEL technique according to the manufacturer's
recommendations (Roche Molecular Biochemicals). Staining was assessed
with peroxidase-conjugated antibody and revealed with a
diaminobenzidine substrate as described (25). Fragmented DNA labeling
corresponds to black spots. A second labeling with erythrosin B was
carried to visualize the totality of the cells.
Caspase 3 Activity--
HEK293 cells were cultured in 6-well
plates and then incubated for 24 h at 37 °C in the presence or
absence of 2.0 µM staurosporine (Sigma). In some cases
cells were either preincubated overnight with 100 µM
Ac-DEVD-al (caspase 3 inhibitor, Neosystem) before treatment with
staurosporine or submitted to a co-incubation of 2 µM
staurosporine and 10 µM p53 inhibitor pifithrin-
.
Cells were then analyzed as extensively described (26). Fluorimetry was
recorded at 360 and 460 nm for excitation and emission wavelengths, respectively, by means of a microtiter plate reader (Labsystems Floroskan II). Caspase-specific activity was calculated from the linear
part of fluorimetry recording and expressed in units/h/mg of proteins
(established by the Bio-Rad procedure). One unit corresponds to 4 nmol
of amidomethylcoumarin released.
Western Blot Analyses--
Plated cells were rinsed, gently
scraped, pelleted by centrifugation, and then resuspended in 100 µl
of lysis buffer (10 mM Tris-HCl, pH 7.5, containing 2% SDS
and a mixture of protease inhibitors, Roche Molecular Biochemicals).
Equal amounts of protein (50 µg) were separated on 8 and 12%
SDS-PAGE gels for analysis of poly(ADP-ribose) polymerase (PARP) and
active caspase 3, Bcl2, caspase 3 precursor (ProCPP32), and p53,
respectively, and wet-transferred to Hybond C (Amersham Biosciences)
membranes. Membranes were then blocked with nonfat milk and incubated
overnight at 4 °C with the following primary antibodies: anti-PARP
(Upstate Biotechnology), anti-active caspase 3 (rabbit polyclonal, R&D
Systems), anti-Bcl2 (mouse monoclonal, Santa Cruz), anti-ProCCP32
(mouse monoclonal, Transduction Laboratories), and an anti-p53 (mouse
monoclonal, Santa Cruz). Immunological complexes were revealed by
enhanced electrochemiluminescence (Roche Molecular Biochemicals) with
either an anti-rabbit peroxidase or with an anti-mouse peroxidase
(Immunotech) antibodies depending on the host used for the production
of the primary antibodies listed above.
Cytochrome c Translocation Analysis--
HEK293 cells were grown
in 6-well plates and incubated for 24 h at 37 °C in the
presence or absence of 2.0 µM staurosporine. Cells were
submitted to a cellular fractionating, and the cytosolic/mitochondrial fractions were electrophoresed on Tris-Tricine gels and
Western-blotted as previously described (13). In brief, 25 µg of
proteins were separated on 16.5 Tris-Tricine gels, immobilized in
nitrocellulose sheets, and probed with an anti-cytochrome c
(rabbit polyclonal, Santa Cruz Technologies) antibody. Immunological
complexes were revealed with an anti-rabbit peroxidase
(Immunotech) and electrochemiluminescence detection (Roche
Molecular Biochemicals).
p53 Transcriptional Activity--
The PG13-luciferase p53 gene
reporter construct (kindly provided by Dr. B. Vogelstein) has been
previously described (27). Cells were cultivated in 12-cell
plates until 70% confluence and then co-transfected with 1.0 µg of PG13-luciferase cDNA and 0.5 µg of a
-galactosidase
transfection vector (to normalize transfection efficiency) by means of
the DAC30 transfection reagent according the manufacturer conditions
(Eurogentec). Forty-eight hours after transfection, luciferase and
-galactosidase activities were analyzed according to manufacturer's
conditions (Promega kit).
Immunoprecipitation and Detection of A
--
HEK293 cells
stably expressing the CTF-PS2 (CPS2) were cultivated in 6-well
plates, preincubated with the inhibitor of caspase 3, Ac-DEVD-al
(Neosystem), for 16 h, and then submitted to a period of 7 h
of secretion in the cultivating media containing 1% instead of 10%
fetal calf serum and 10 µM phosphoramidon (Sigma). Media were then recovered and analyzed for total A
by combined
immunoprecipitation FCA18 (28) and Western blot with WO2 as previously
described (29).
 |
RESULTS |
The setting up and characterization of stably HEK293 cells
overexpressing the CTF of PS2 was previously reported (24). We previously showed that overexpression of the CTF-PS2 in these cells
(compare lanes without Ac-DEVD-al in Fig.
1C) led to an increased
recovery of total amyloid
-peptides (24). Here we confirm that
CTF-PS2-expressing HEK293 cells secrete more A
than mock-transfected
control cells in basal conditions (Fig. 1, A and
B). Interestingly, the caspase 3 inhibitor Ac-DEVD-al
drastically reduces A
recovery in both cell systems (Fig. 1,
A and E) and concomitantly increases endogenous
(MOCK) and overexpressed (CPS2) CTF-PS2-like
immunoreactivity (Fig. 1, C and F). Conversely,
CTF-PS2 immunoreactivity was drastically lowered by the treatment of
transfected cells with the apoptotic stimulus staurosporine (Fig.
1D) in an Ac-DEVD-al-sensitive manner (not shown). This was
accompanied by a staurosporine-stimulated increase of A
recovery in
both mock- and CTF-PS2-transfected cells (Fig. 1B). This
modulation of the CTF-PS2-mediated control of
APP maturation by
apoptotic effector and inhibitor led us to deeply examine the putative
role of CTF-PS2 in the control of cell death.

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Fig. 1.
CTF-PS2 increases A
recovery; effect of caspase 3 inhibitor (Ac-DEVD-al) and
staurosporine. Indicated cells were pretreated overnight with (+)
or without ( ) 100 µM Ac-DEVD-al (A) then
total secreted A was analyzed by immunoprecipitation and Western
blot as depicted under "Materials and Methods" and quantitated by
densitometry (E). CTF-PS2-like immunoreactivity in both cell
lysates was analyzed (C) and quantitated in CPS2 cells
(F) by densitometry. Bars in E and
F are the means ± S.E. of five independent experiments
(n = 5). In B, the indicated cells were
incubated for the indicated time periods with 2 µM
staurosporine (STS) and analyzed for their A production.
CTF-PS2-like immunoreactivity in D corresponds to a 24-h
treatment with 2 µM staurosporine.
|
|
First, we examined the capacity of CTF-PS2 to alter cell viability by
means of TUNEL and DNA fragmentation analyses. TUNEL of apoptotic cells
shows that overexpression of CTF-PS2 increases the number of stained
nuclei in both basal and apoptotic conditions when compared with
mock-transfected cells (Table I).
Fluorescence-activated cell sorter analysis of propidium iodide nuclei
incorporation also showed increased DNA fragmentation in basal and
staurosporine-stimulated cells (Table I). These two CTF-PS2-mediated
phenotypes clearly illustrate a pro-apoptotic phenotype underlying
associated decrease in cell viability.
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Table I
CTF-PS2 triggers DNA fragmentation and increases the number of
tunel-positive cells
Apoptosis and DNA fragmentation were monitored by Tunel and
fluorescence-activated cell sorter analyses as described under
"Materials and Methods" in mock- or CTF-PS2-expressing cells in the
absence (basal) or in presence of staurosporine. Means ± S.E.
correspond to three independent experiments carried out in duplicate.
|
|
Another signature of such an apoptotic process is the drastic increase
of the Ac-DEVD-al-sensitive caspase 3-like activity triggered by
CTF-PS2 in both basal and staurosporine-induced conditions (Fig.
2A). In agreement, Fig.
2B shows a drastic reduction of the immunoreactivity of the
pro-caspase 3 precursor in cells overexpressing CTF-PS2, reflecting an
increased maturation of the inactive precursor into its bioactive
counterpart in CTF-PS2 transfected cells. We further examined caspase 3 modulation through the monitoring of the cleavage of one of its favored
substrate, the poly-(ADP-ribose) polymerase (PARP). It is well
documented that PARP occurs as an inactive precursor that is
proteolytically converted into its active counterpart by caspase 3. Thus the ratio between PARP precursor over its product is generally an
index of caspase 3 activity. In mock-transfected cells, as expected,
the apoptotic stimulus staurosporine increases PARP product (Fig.
3A) and, therefore, reduces
the above-described ratio (Fig. 3B). Here again, cells expressing CTF-PS2 are associated with increased production of PARP
product in both basal and staurosporine-stimulated conditions (Fig.
3A), resulting in a drastically lower ratio when compared with mock-transfected cells, which indirectly again reflects increased caspase 3 activity (Fig. 3B).

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Fig. 2.
CTF-PS2 increases caspase 3-like
activity. A, basal (black bars) and
STS-stimulated (2 µM staurosporine (STS),
24 h, white bars) caspase 3-like activity was
fluorimetrically recorded in the indicated cell lines as described
under "Materials and Methods." Bars represent the
Ac-DEVD-al-sensitive caspase 3-like activity and are the means ± S.E. of eight determinations carried out in duplicates. B,
determination of Pro-CPP32-like immunoreactivity (inset) and
quantitative densitometric analysis in basal conditions in the
indicated cell lines. The histograms represent the quantitative
analysis of five independent determinations expressed in % (means ± S.E.) of control mock-transfected cells taken as 100.
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Fig. 3.
CTF-PS2 overexpression increases PARP
cleavage in HEK293 cells. A, a representative gel of
the PARP precursor (Prec) cleavage into its product
(Prod) performed in basal (CT) and stimulated
(STS) conditions (2 µM STS, 24 h) in
mock- or CTF-PS2-expressing cells. The histograms in B
represent the quantitative analysis of five independent determinations
expressed as the ratio precursor/product densitometric analysis
reflecting PARP activation.
|
|
To delineate the mechanisms by which CTF-PS2 could exert its caspase
3-mediated pro-apoptotic phenotype, we examined the putative contribution of the mitochondrial pathway. Staurosporine increases the
translocation of cytochrome c from the mitochondrial
fraction to the cytosol in mock-transfected cells (Fig.
4, A and B).
Overexpression of CTF-PS2 triggers a significant increase of the
translocation of cytochrome c (Fig. 4, A and
B) associated with reduced immunoreactivity of the
antiapoptotic oncogene Bcl2 (Fig. 5,
A and B), whereas Bax-like immunoreactivity
remained unaffected (not shown). This likely contributed to the
perturbation of the mitochondrial membrane permeability, resulting in
cytochrome c translocation. It should be noted that in
agreement with these data, CTF-PS2-expressing cells show a 250 and
175% increase in propidium iodide incorporation in basal and
stimulated conditions, respectively, after cell treatment with
etoposide, an apoptotic effector targeting the mitochondrial compartment (not shown).

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Fig. 4.
CTF-PS2 increases cytochrome c
translocation to the cytosol. A, the indicated
cell lines were treated (STS) or not (C) with
staurosporine (2 µM, 24 h) and analyzed for their
cytochrome c content in cytosolic (c) and
mitochondrial (m) fractions obtained as detailed under
"Materials and Methods." Bars in B correspond
to the means ± S.E. of five independent experiments and represent
the ratios of cytochrome c immunoreactivities in cytosol
versus mitochondrial fractions.
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Fig. 5.
CTF-PS2 overexpression decreases Bcl2-like
immunoreactivity. A, determination of Bcl2 expression
in the indicated cell lines as detailed under "Materials and
Methods" and their quantitative densitometric analysis in
B. Bars represent five independent determinations
(means ± S.E.) expressed in percent of control mock-transfected
cells taken as 100.
|
|
We examined whether CTF-PS2 expression could elicit its pro-apoptotic
phenotype by a p53-dependent mechanism. As shown in Fig.
6A, stably transfected CPS2
cells show an increased level of p53 immunoreactivity that is virtually
doubled in transfected cells (Fig. 6B). This increased
immunoreactivity is accompanied by a drastic potentiation of p53
transcriptional activity (Fig. 6C).

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Fig. 6.
CTF-PS2 increases p53 expression and
transcriptional activity. A, determination of p53
immunoreactivity in basal (CT) and staurosporine
(STS)-stimulated conditions (2 µM STS, 24 h) in the indicated cell lines. Bars in B
represent the densitometric analysis of p53-like immunoreactivity and
are the means ± S.E. of three independent determinations.
C, determination of p53 transcriptional activity (see
"Materials and Methods") measured with a p53 gene reporter
(PG13-luciferase). Bars represent the means ± S.E. of
three independent experiments made in duplicate.
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We took advantage of the recent description of pifithrin-
, a
selective blocker of p53 transcriptional activity to show a direct
relationship between CTF-PS2-mediated p53 and caspase 3 activations.
Fig. 7 shows little if any effect of
PTF
in mock-transfected cells examined in basal conditions, whereas
about 50% of the caspase 3 activity was inhibited in
CTF-PS2-expressing cells (compare black and white
bars in Fig. 7), indicating that the higher caspase3 activity
observed in basal conditions in the latter cell system (Fig.
2A) was related to p53 activity. An identical blockade of caspase 3 activation was observed in staurosporine-stimulated conditions (Fig. 7), indicating that both basal and
staurosporine-induced caspase activation triggered by CTF-PS2
expression were blocked by PTF
. This demonstrates the direct
modulation of caspase 3 activity by p53 as the main mechanism
underlying CTF-mediated proapoptotic phenotype.

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Fig. 7.
Effect of pifithrin-
on CTF-PS2-induced activation of caspase 3. The indicated
cell lines were treated for 24 h in the presence or absence of 10 µM pifithrin- (PFT) in basal and
staurosporine-stimulated conditions (2 µM, STS).
Bars represent the means ± S.E. of three independent
fluorimetric analysis (duplicates) of caspase 3 activity expressed in
percent of control (taken as 100) corresponding to untreated
cells.
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Finally, to examine whether the CTF-PS2 was able to elicit its
pro-apoptotic function in absence of its presenilinase-derived N-terminal counterpart, we transiently transfected fibroblasts in which
the genes coding for PS1 and PS2 were invalidated with CTF-PS2
cDNA. Analyses of caspase 3-like activity in basal and STS-induced
apoptosis clearly shows that the CTF-PS2 remains proapoptotic in
PS
/
fibroblasts (Fig.
8C). It is noteworthy that the
effect of CTF-PS2 was significant only in stimulated conditions (Fig.
8C). This could be explained by the relatively low
transfection efficiency observed (Fig. 8A). Accordingly, the
immunoreactivity of active caspase 3 (Fig. 8B) was enhanced
in staurosporine-treated CTF-PS2-transfected PS
/
fibroblasts.

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Fig. 8.
CTF-PS2 increases caspase 3 activity in
PS1 / PS2 /
mouse embryonic fibroblasts. A, CTF-PS2-like
immunoreactivity after its cDNA transient transfection.
B, two independent determinations of active caspase 3 immunoreactivity by Western blot analysis in basal (Ct) and
staurosporine (STS)-stimulated conditions (1 µM, 2 h) in PS / -deficient cells
transiently transfected either with empty vector (pcDNA3) or
CTF-PS2 cDNA as detailed under "Materials and Methods."
Bars in C correspond to caspase 3 fluorimetric
activity and are the means three independent experiments
performed in duplicate in basal and stimulated (STS)
conditions (STS, 1 µM, 2h). Data were normalized for
CTF-PS2 transfection efficiency assessed by Western blot as described
under "Materials and Methods". ns, not
significant.
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|
Additionally, these results show that CTF-PS2 triggers the same
phenotype in cell types of different origin, namely HEK293 human cells
(Figs. 2-7), mouse embryonic fibroblasts (Fig. 8), and telencephalon-specific murine (TSM1) neurons (data not shown), indicating that its pro-apoptotic function is not cell-specific.
 |
DISCUSSION |
PS are multipotent proteins likely involved in distinct functions
including among others control of
APP processing, Notch and Wnt
pathways, and adhesion (5). PS1 and PS2 also appear to be involved in
the modulation of cell death, although the two parent proteins seem to
behave differently in this paradigm. Thus, PS2 was clearly
characterized by several groups as a proapoptotic effector (13,
30-33), whereas PS1 decreases the susceptibility of neurons to
apoptotic stimuli (34). It is interesting to note that although the two
proteins elicit opposite phenotypes, they both appear to be linked to
the tumor suppressor p53 activity. Thus, it was established that p53
down-regulated PS1 expression, thereby leading to delayed apoptotic
response (14). By contrast, we recently showed that PS2 and its
pathogenic mutant N141I-PS2 drastically increased p53 expression and
transcriptional activity in various cell systems (13). Interestingly,
this was accompanied by a reduction of PS1 expression, suggesting a
cross-talk between the two congener proteins (13).
PS are rapidly processed in the endoplasmic reticulum, leading to
N-terminal (NTF-PS) and C-terminal (CTF-PS) fragments that appear to
associate with a 1/1 stoichiometry (7). This heterodimer is thought to
correspond to the biologically active complex responsible for
PS-associated functions. Whether the maturated fragments could per se modulate PS-related functions remained a matter of
speculation. However, a pivotal work by Vito et al. (35)
indicates that ALG3, the C-terminal 103 amino acids of mouse PS2, was
able to rescue a T cell hybridoma from Fas-induced apoptosis (35).
Furthermore, the same group showed that a C-terminal fragment of PS2
that can be physiologically generated by proteolysis and alternative
transcription was able to rescue HeLa cells from Fas- and tumor
necrosis factor-induced apoptosis (36). These studies could be
envisioned as clues of functions specifically associated with
PS-derived maturated fragments, although it could be argued that
overexpression of these products displace their endogenous counterparts
from the biological heteromeric complex. This possibility would not
explain how the sole expression of one fragment could trigger a
phenotype if this just results in a substitution limited
stoichiometrically by the other endogenous counterpart.
We previously showed that CTF-PS1 and CTF-PS2 overexpression led to an
up-regulation of
APP processing, yielding increased amounts of both
- and
/
-derived products APP
and A
, respectively (24).
We confirm that A
production is increased upon overexpression of
CTF-PS2 in the present study. This phenotype appears blocked by caspase
3-like inhibitors and stimulated by staurosporine with accompanying
increase or lowering of CTF-PS2-like immunoreactivity, respectively.
This suggests that increased A
production is related to the
caspase-derived cleavage product of CTF-PS2. The fact that CTF-PS2
undergoes breakdown upon staurosporine stimulus also argues for the
possibility that CTF-PS2 behaves as the precursor of a caspase-derived
fragment that could control cell death. This would well agree with the
study carried out by Walter et al. (32) that showed that
phosphorylation of PS2 slowed down caspase-mediated cleavage and
retarded apoptosis in HeLa cells.
The mechanisms by which CTF-PS2 could trigger cell death remained to be
established. Here we show for the first time that CTF-PS2
overexpression led to increased caspase 3 activity and immunoreactivity, enhanced cleavage of one of the favorite caspase 3 substrates, poly(ADP-ribose) polymerase, and decreased expression of
the antiapoptotic oncogene bcl-2. Whether the physical
interaction between CTF-PS2 with bcl-2 described by Passer
et al. (37) could explain our observed increased
translocation of cytochrome c in the cytosol remains to be
established. We established that the CTF-PS2-associated cell death was
a p53-dependent phenomenon. Thus, CTF-PS2 increases both
p53 expression and transcriptional activity (see Fig. 7). That
CTF-PS2-associated p53 modulation and caspase activation were directly
linked was evidenced by the fact that pifithrin-
, a selective
blocker of p53, fully prevents the CTF-PS2-induced caspase activation
(see Fig. 8). The mechanisms by which CTF-PS2 triggers cell death
appear similar to the one we described for PS2 itself. Therefore, the
possibility that CTF-PS2-associated phenotype could account entirely
for that observed with PS2 remains a possibility.
Interestingly, we demonstrate that CTF-PS2 also triggers caspase 3 activation in cells devoid of presenilins. Thus, PS
/
cells respond to transient transfection with CTF-PS2 by an augmentation of both caspase 3 immunoreactivity and activity. This means that unlike
what appears to be the case for APP processing, the presence of the
N-terminal counterpart is not an absolute requirement for CTF-PS2
proapoptotic phenotype. It is interesting to note that CTF-PS2-like
immunoreactivity could be detected in the liver of adult mice in which
PS genes had been disrupted (35, 38). This observation combined with
the present work could suggest possible functions specifically
associated with CTF-PS2 in absence of its presenilinase-derived
N-terminal counterpart.
 |
ACKNOWLEDGEMENTS |
We thank Drs. T. Hartmann and K. Beyreuther
for WO2 monoclonal antibodies. We are grateful to Drs. P. Saftig
and B. de Strooper for providing PS
/
fibroblasts. Dr.
Bert Vogelstein kindly provided the PG13-luciferase construct.
 |
FOOTNOTES |
*
This work was supported by the INSERM and CNRS.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.
§
Supported by Aventis Pharma.
To whom correspondence should be addressed: Intitut de
Pharmacologie Moléculaire et Cellulaire, UMR6097 du CNRS, 660 Route des Lucioles, Sophia Antipolis, 06560 Valbonne, France. Tel.: 33-493957760; Fax: 33-493957708; E-mail: checler@ipmc.cnrs.fr.
Published, JBC Papers in Press, January 29, 2003, DOI 10.1074/jbc.M212379200
 |
ABBREVIATIONS |
The abbreviations used are:
PS, presenilin;
CTF, C-terminal fragment;
PARP, poly(ADP-ribose) polymerase;
HEK
cells, human embryonic kidney cells;
Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl] glycine;
TUNEL, terminal dUTP nick-end labeling;
Ac-DEVD-al, acetyl-DEVD-aldehyde.
 |
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