From the Dipartimento di Scienze e Tecnologie
Biomediche, Sezione di Biologia, Universita' di Udine, P. le Kolbe 4, Udine 33100, Italy and ¶ Laboratorio Nazionale Consorzio
Interuniversitario Biotecnologie AREA Science Park, Padriciano 99, Trieste 34142, Italy
Received for publication, December 21, 2000, and in revised form, March 14, 2001
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ABSTRACT |
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Mammalian caspases are a family of cysteine
proteases that plays a critical role in apoptosis. We have analyzed
caspase-2 processing in human cell lines containing defined mutations
in caspase-3 and caspase-9. Here we demonstrate that caspase-2
processing, during cell death induced by UV irradiation, depends both
on caspase-9 and caspase-3 activity, while, during
TNF- Caspases belong to a conserved family of cysteine proteases
playing a critical role in apoptosis and proinflammatory cytokine maturation (1). In normal cells, caspases are present as zymogens that
are cleaved at aspartic sites during cell death. Enzyme processing generates the active form, which is constituted by a heterodimer consisting of two small and a two large subunits (2).
Caspases involved in the apoptotic process can be divided into
initiator caspases and effector caspases based on the presence of a
large prodomain at their amino terminus region. In general, effector
caspases possess short prodomains and are cleaved by initiator
caspases, which possess large prodomains. Effector caspases are
involved in the cleavage of the death substrates, thus modulating the
morphological changes characterizing the apoptotic process (3).
The long prodomains of the initiator caspases function in inducing
dimerization and activation of the proenzymes through interaction with
specific adaptor molecules. Caspase-2, caspase-8, caspase-9, and
caspase-10 are the long prodomain caspases involved in the apoptotic
process (4).
The generation of caspase-deficient mice for initiator caspases has
indicated that these enzymes regulate cell death in a tissue- and
stimulus-specific fashion (4). In particular, caspase-9 is the critical
player of the apoptotic stimuli acting through mitochondrial
dysfunction (5, 6), while caspase-8 is critical for the apoptotic
pathways generated by death receptors (7).
Caspase-2-deficient mice showed an apoptotic deficit in the oocytes,
following exposure to chemotherapeutic drugs, and in B lymphoblasts
following incubation with perforin and granzyme B (8). Additional
studies have shown that this initiator caspase is essential for
specific apoptotic pathways such as The identification of RAIDD/CRADD as an adaptor protein for caspase-2
has suggested that caspase-2 could be involved also in apoptosis
triggered by TNF- Different apoptotic insults can cause caspase-2 processing, which
generally occurs by two proteolytic steps. A first cleavage at aspartic
316 generates two fragments: one of 32-33 kDa containing the prodomain
and the large subunit and a second fragment of 14 kDa containing the
small subunit. Subsequent cleavages at Asp152 and
Asp330 lead to the formation of the large and the small
subunits of 18 and 12 kDa, respectively. The appearance of the
32-33-kDa fragment has been generally used as marker of caspase-2
activation (13-15).
Despite significant evidence for an involvement of caspase-2 in
different apoptotic pathways, its specific role is not completely understood. Only recently, a caspase-2 substrate has been identified in
golgin-160, thus suggesting that caspase-2 could act as an executioner
caspase involved in modulating Golgi integrity (16).
In the present study, we have attempted to further analyze caspase-2
activity and its hierarchy with respect to the common apoptotic
pathways. We have used human cell lines containing defined mutations in
caspase-9 and caspase-3 to unveil the relationships between the
mitochondrial pathway and caspase-2 proteolytic processing in response
to different apoptotic triggers. In addition, we have analyzed the
ability of caspase-2 to induce cell death in cells defective in
caspase-9 activity.
Antibody Production--
Rabbits were immunized with His-tagged
caspase-2 fragment (residues 15-436) purified from Escherichia
coli transformed with the construct pQE32-caspase-2. Briefly,
after induction with
isopropyl-1-thio-
Purified recombinant caspase-2 was cross-linked to an Affi-Prep 10 column (Bio-Rad) and used to affinity-purify antibody to caspase-2 from
rabbit antiserum. The serum was loaded onto the column at a slow flow
rate, washed with 10 mM Tris-HCl, pH 7.4, and then washed
with a high salt buffer (500 mM NaCl, 10 mM
Tris-HCl, pH 7.4). Caspase-2 antibodies were eluted using 10 mM glycine, pH 2.5, and neutralized with pH 7.5 with
Tris-HCl.
Plasmids--
To generate pcDNA3HA caspase-2
C303G, the entire coding region of human caspase-2 was amplified from
pGDSV7S caspase-2 by polymerase chain reaction.
In vitro mutagenesis to substitute Cys303 with
Gly was performed as previously described (17). The following set of
primers was used: primer A (CATGAATTCATGGCCGCTGACAGGGGACGC),
primer B (ATCCAGGCCGGCCGTGGAGAT), primer C (ATCTCCACGGCCGGCCTGGAT), and primer D (CATCTCGAGTCATGTGGGAGGGTGTCCTGG).
Caspase-2 C303G cDNA was subcloned in pcDNA3HA as
EcoRI/EcoRI and EcoRI/XhoI fragments.
Caspase-2 C303G was subcloned from pcDNA3HA caspase-2
C303G as SphI/XhoI fragment in pQE32 to generate
a His-tagged caspase-2 construct.
To generate pEGFPN1Bid and pBSKBid, the entire coding region of human
Bid was amplified by polymerase chain reaction from a human fetal
cDNA library. The following set of primers was used: primer
E (CATGAATTCTGATGGACTGTGAGGTCAACAAC) and primer F (CATGGATCCCGGTCCATCCCATTTCTGGCTAA).
Cell Lines and Induction of Apoptosis--
Cells were grown in
Dulbecco's modified Eagle's medium supplemented with 10% fetal calf
serum, penicillin (100 units/ml), and streptomycin (100 µg/ml).
Cells at 70-80% confluence were treated for 36 h with 1 µg/ml
cycloheximide plus 20 ng/ml TNF-
For UV treatment, culture medium was removed, dishes were washed once
with PBS1 and UVC (180 J/m2)-irradiated, and fresh medium, containing 10% fetal
calf serum, was added to the cells (17). Cells were harvested by
scraping with a rubber policeman 15 or 18 h later. When required
floating cells were collected separately from the adherent cells. After washes in PBS, cells were resuspended in SDS sample buffer sonicated boiled for 3 min and analyzed by Western blot.
Transfections were performed by the calcium phosphate precipitation
method. Cell were seeded 24 h before transfection and analyzed
20 h after removal of the precipitates. Cells were transfected with 2 µg of each expression vector together with 200 ng of pEGFPN1 (Invitrogen) to identify transfected cells.
In Vitro Proteolytic Assay--
Caspase-3 was expressed in
bacteria and purified as previously described (18) using the pQE-12
expression system (Qiagen). Density scanning of the ~20-kDa fragments
of the autoprocessed caspases, as evidenced after electrophoretic
separation and Coomassie Blue staining, was used to estimate the amount
of active enzyme. Purified caspase-7 was obtained from Alexis, and
caspase-2 was from Chemicom.
The different caspases, poly(ADP-ribose) polymerase (PARP) and Bid,
were in vitro translated with 35S using the
TNT-coupled reticulocyte lysate system (Promega). 1 µl of each
in vitro translated protein was incubated with increasing amounts of caspase-3 or caspase-7 in 15 µl of the appropriate buffer
(final volume) for 1 h at 37 °C. Reactions were terminated by
adding one volume of SDS gel loading buffer and boiling for 3 min.
Immunoblotting--
For Western blotting, proteins were
transferred to 0.2-µm pore-sized nitrocellulose (Schleicher & Schuell) using a semidry blotting apparatus (Amersham Pharmacia
Biotech) (transfer buffer: 20% methanol, 48 mM Tris, 39 mM glycine, and 0.0375% SDS). After staining with Ponceau
S, the nitrocellulose sheets were saturated for 1 h in
Blotto-Tween 20 (17) (50 mM Tris-HCl, pH 7.5, 200 mM NaCl, 5% nonfat dry milk, and 0.1% Tween 20) and
incubated overnight at room temperature with the specific antibody:
anti-caspase-2, anti-actin, and anti-p85 PARP fragment (Promega). Blots
were then rinsed three times with Blotto-Tween 20 and incubated with
peroxidase-conjugated goat anti-rabbit (Sigma) or goat anti-mouse
(Sigma) for 1 h at room temperature. The blots were then washed
four times in Blotto-Tween 20, rinsed in phosphate buffer saline, and
developed with Super Signal West Pico, as recommended by the vendor (Pierce).
Indirect Immunofluorescence Assay--
For indirect
immunofluorescence assays, transfected cells were fixed with 3%
paraformaldehyde in PBS for 1 h at room temperature. Fixed cells
were washed with PBS and 0.1 M glycine, pH 7.5, and then
permeabilized with 0.1% Triton X-100 in PBS for 5 min. The coverslips
were treated with anti-cytochrome c (Promega) or with anti-cytochrome c oxidase, diluted in PBS for 1 h in a
moist chamber at 37 °C. They were then washed with PBS twice,
followed by incubation with tetramethylrhodamine
isothiocyanate-conjugated anti-mouse (Sigma) for 30 min at 37 °C.
Nuclei were evidenced by Hoechst staining.
Cells were examined by epifluorescence with a Zeiss Axiovert 35 microscope or with a LEICA TCS laser scan microscope equipped with a
488 Production of a Caspase-2-specific Antibody--
We produced an
antibody against E. coli expressed caspase-2 fragment in
order to study caspase-2 activation during cell death. The antiserum
was purified on a caspase-2 affinity column and tested for caspase-2
detection by Western blot. 293 cells were transfected with HA-tagged
catalytic inactive caspase-2 as positive control. Anti-caspase-2
antibody recognizes a single band migrating at around 48 kDa, showing a
similar electrophoretic mobility to the band detected with the anti-HA
antibody in caspase-2-transfected cells. The intensity of the band
detected by the anti-caspase-2 antibody was dramatically increased in
caspase-2-transfected cells (Fig.
1a).
Caspase-2 processing during apoptosis can be followed by the appearance
of different proteolytic fragments. A first cleavage at aspartic 316 generates two fragments: a first fragment of 32-33 kDa containing the
prodomain and the large subunit and a second fragment of 14 kDa
containing the small subunit. Subsequent cleavages at
Asp152 and Asp330 generate the large and the
small subunits of 18 and 12 kDa, respectively (13). To confirm the
specificity of the anti-caspase-2 antibody, we investigated caspase-2
processing by Western blot during apoptosis.
MDA cells were UV-irradiated, and cellular lysates were prepared
24 h later. In UV-irradiated cells, a band migrating at ~33 kDa
was detected, the appearance of which parallels the rate of cell death
in the culture as confirmed by
We next investigated the appearance of the 33-kDa form of
caspase-2 in different cell lines when apoptosis was induced by UV
irradiation. As shown in Fig. 1c, the processed form of
caspase-2 was detected upon apoptosis induction in most of the cell
lines tested, with the exception of the MCF-7 cells. The anti-caspase-2 antibody also showed cross-species reactivity, since it detected a band
of 50 kDa in the cellular lysates of murine fibroblasts NIH 3T3 that is
converted in a p33 form upon induction of apoptosis (data not shown).
IMR90-E1A Containing a Dominant Negative Form of Caspase-9 and
MCF-7 Containing a Catalytically Inactive Caspase-3 Show a Defective
Apoptotic Response following UV Irradiation--
To study the
hierarchy of caspase-2 processing in respect to different caspases
during apoptosis in vivo, we decided to use cell lines
containing defined mutations in specific caspases.
We took advantage of the IMR90 cells transformed with E1A oncogene and
containing a dominant negative form of caspase-9 (caspase-9 DN) (20).
As a control IMR90 fibroblasts expressing E1A alone were used. It has
been previously demonstrated that IMR90-E1A cells expressing the
caspase-9 DN do not show all of the "classical" apoptotic features
when challenged by etoposide treatment, whereas in these cells
cytochrome c release from mitochondria was reported as
normal (20).
We also used the human MCF-7 breast carcinoma cell line, which is
devoid of caspase-3 due to the functional deletion of the CASP-3 gene (21). This cell line was used to
reintroduce wild type caspase-3 or its catalytic inactive point-mutated
derivative caspase-3 (CI) as a control (22).
We used FACS analysis to confirm that the different cell lines selected
showed different susceptibility to enter cell death by apoptosis
following UV irradiation.
UV-irradiated IMR90-E1A cells underwent apoptosis as evidenced by cell
morphology, chromatin condensation, and detachment from the adhesion
substrate. Levels of apoptosis were assessed by FACS analysis using
propidium iodide staining (Fig.
2a). Analysis of DNA content
(Fig. 2) revealed a significant increase of cells with
sub-G1 DNA content, indicative of apoptosis (19), when IMR90-E1A cells were UV-irradiated. On the contrary, IMR90-E1A cells
containing caspase-9 DN were impaired in entering efficiently apoptosis
following UV irradiation, as evidenced by FACS analysis. Indeed, in
these cell lines, floating cell fragments could be detected upon UV
irradiation (data not shown).
Reintroduction of wild type caspase-3 in MCF-7 cells renders these
cells susceptible to apoptosis following UV irradiation, whereas cells
expressing a catalytic inactive form of caspase-3 were partially
resistant to UV-dependent cell death (Fig. 2b). Moreover, when MCF-7 caspase-3 CI cells were UV-irradiated, some cell
debris was detected floating in the medium (data not shown).
Caspase-2 Processing in Human Fibroblasts Expressing a Catalytic
Inactive Form of Caspase-9--
Having selected two human cell lines
containing defined mutations in caspase-3 and caspase-9, we analyzed
the relationships between caspase-2 processing and the above mentioned
caspases by inducing cell death with two different stimuli: UV
irradiation and TNF-
As mentioned before, some dead cells floating in the medium were
observed also in the case of UV-irradiated IMR90-E1A-caspase-9 DN that
were resistant to apoptosis as judged by FACS analysis. These dead
cells might be indicative of a necrotic or "frustrated apoptotic"
response that occurs following extensive DNA damage and/or
mitochondrial dysfunction.
In our studies, we have focused our attention on the status of
caspase-2 in the population of dead cells that can be isolated as
nonadherent cells (Fig. 3, D)
from the population of still viable cells (Fig. 3, V). It is
important to note that the population of nonadherent cells was
consistently reduced in cells expressing the catalytic inactive
caspases (data not shown), but in our Western analysis the total amount
of protein lysates loaded for each sample was normalized.
In apoptotic IMR90-E1A cells, almost all caspase-2 was processed to the
p33 form, as shown in Fig. 3a, while in floating cells from
IMR90-E1A-caspase-9 DN (D), caspase-2 processing was largely impaired. The same lysates were also analyzed for PARP processing by
using an antibody specific for the cleaved form. Processing of PARP was
observed in both cell lines although the presence of a dominant
negative form of caspase-9 clearly reduced the amount of cleaved PARP
detectable. Actin was used as loading control.
We next analyzed the dependence of caspase-2 processing on caspase-9 in
response to the activation of a different apoptotic pathway. IMR90-E1A
and IMR90-E1A-caspase-9 DN were treated with TNF-
In TNF- Caspase-2 Processing during Apoptosis in MCF-7 Cells--
The
previous experiment clearly shows that caspase-2 processing following
UV irradiation is dependent on caspase-9. Since caspase-9 activates
caspase-3 in response to DNA damage (20), we next analyzed if caspase-3
is critical for caspase-2 processing in vivo.
Human MCF-7 cells were UV-irradiated, and 18 h later nonadherent
dead cells (Fig. 3, D) and adherent viable (V)
cells were harvested and combined, and lysates were prepared for
Western analysis.
As shown in Fig. 4a, in
UV-irradiated MCF-7 wild type caspase-3, a consistent fraction of
caspase-2 could be detected as the p33-processed form, while in MCF-7
caspase-3 CI cells, caspase-2 processing was undetectable. Processing
of PARP was reduced but still detectable in MCF-7 caspase-3 CI as
described above in IMR90-E1A caspase-9 DN.
We next analyzed the dependence of caspase-2 processing on caspase-3
under TNF-
Caspase-2 was fully processed when apoptosis was induced in wild type
caspase-3 MCF-7 cells treated with TNF-
PARP was similarly processed in both cell lines, thus confirming the
induction of cell death.
In Vitro Caspase-3-mediated Processing of Caspase-1, -2, -3, -6, -7, -8, and -9--
Having demonstrated that caspase-3 is the critical
enzyme for an efficient processing of caspase-2 in vivo, we
wanted to analyze the ability of caspase-3 to cleave caspase-2 in
vitro with respect to other long and short prodomain caspases
involved in the apoptotic response.
In vitro proteolytic assays using recombinant caspases-3
were performed. Full-length caspase-1, caspase-2, caspase-3, caspase-6, caspase-7 caspase-8, and caspase-9 cDNA were in vitro
translated and then incubated with purified caspase-3 (Fig.
5).
Treatment with an increasing amount of purified caspase-3 for 30 min at
37 °C leads to the processing of all of the tested caspases with the
exception of caspase-1. Among the different long prodomain caspases,
some processing of caspase-2 to generate the p18/p12 active form was
detectable after incubation with only 0.01 ng of caspase-3, while the
full processing of the p48 form was detectable when 500 ng of caspase-3
were used. In contrast, caspase-9 and caspase-8 were weak substrates of
caspase-3 and only partially processed after incubation with 10 ng of
the recombinant caspase-3. Among the different short prodomain caspases
analyzed, caspase-7 was the most efficiently cleaved by caspase-3, and
partial processing was detectable after incubation with 10 ng of
recombinant caspase-3.
PARP, a well defined substrate of caspase-3 (4, 23), was used as
a control under the same conditions. From this analysis, we can suggest
that caspase-2, among the different caspases analyzed, is the best
substrate for caspase-3.
In Vitro Processing of Caspase-2 Can Be Mediated by
Caspase-7--
Residual caspase-2 processing was observed when
apoptosis was induced by TNF-
In vitro translated caspase-2 was incubated with increasing
amounts of purified caspase-7 for 60 min at 37 °C. As shown in Fig.
6a, 1 µg of caspase-7 was
required for the full processing of caspase-2, while partial caspase-2
processing was observed after incubation with 10 ng of caspase-7. Under
the same experimental conditions, full processing of PARP was observed
after incubation with 10 ng of purified caspase-7. This analysis
suggests that caspase-7 can cleave caspase-2 in vitro,
although with a lower efficiency when compared with PARP.
It is possible that caspase-7 is responsible for the limited
proteolytic processing of caspase-2 during apoptosis in MCF-7 caspase-3
CI cells (25). We therefore analyzed if caspase-7 was activated when
these cells were treated with TNF-
We have analyzed by Western blot caspase-7 expression, as shown in Fig.
6b, with an antibody that recognizes the caspase-7 proenzyme
migrating at around 34 kDa. Disappearance of the 34-kDa band was
considered as evidence for caspase-7 processing. Nonadherent apoptotic and adherent nonapoptotic cells were harvested separately.
Caspase-7 was processed in MCF-7 wild type caspase-3 cells during
apoptosis, and its processing was reduced but still detectable in
apoptotic MCF-7 containing catalytic inactive caspase-3.
Apoptosis Induced by Caspase-2 Overexpression Triggers Cytochrome c
Release and Requires Caspase-9 Activity--
The dependence of
caspase-2 processing on caspase-3 argues that caspase-2 acts as an
effector caspase and that its activation is a downstream event in the
proteolytic cascade triggering cell death.
On the other hand, caspase-2 possesses a prodomain, which is a marker
for caspases acting at the apex of the proteolytic cascade, and its
overexpression can trigger cell death by apoptosis, possibly as a
consequence of caspase-2 activation following prodomain-mediated oligomerization (14, 15, 26, 27).
If caspase-2 induces apoptosis simply by acting as an effector caspase,
its ability to induce cell death should be independent from mutations
in the regulative caspases such as caspase-9.
Therefore, we have investigated the ability of caspase-2 to
efficiently induce cell death when overexpressed in cells impaired in
caspase-9 activity.
IMR90-E1A caspase-9 DN and IMR90-E1A were co-transfected with caspase-2
and GFP used as a reporter, and the appearance of apoptotic cells was
scored 24 h after transfection.
As shown in Fig. 7a, the
apoptotic response triggered by caspase-2 overexpression in IMR90-E1A
caspase-9 DN was impaired, thus demonstrating the dependence on
caspase-9. Under the same conditions, PLAP (placental alkaline
phosphatase), used as a control, was unable to induce cell death.
The dependence of caspase-2 from caspase-9 to efficiently induce
apoptosis was similarly observed in the case of Bax, which is a well
known modulator of the mitochondrial integrity (28).
Caspase-9 is part of the apoptosome that triggers cell death in
response to mitochondrial stress and subsequent release of mitochondrial components such as cytochrome c (29-32).
Caspase-2 in turn requires caspase-9 to mediate apoptosis, therefore
suggesting that caspase-2 acts upstream of the mitochondrial pathway.
Therefore, we next analyzed by immunofluorescence whether overexpression of caspase-2 leads to cytochrome c release
from mitochondria.
As exemplified in Fig. 7b, IMR90-E1A caspase-9 DN cells
overexpressing caspase-2 showed relocalization of cytochrome
c, as a diffused staining in the cytoplasm (see
arrows). In nonoverexpressing cells, cytochrome c
showed a filamentous, perinuclear distribution, an indication for
mitochondrial localization. Fig. 7c shows a quantitative
analysis of cytochrome c localization in IMR90-E1A and
IMR90-E1A caspase-9 DN cells overexpressing caspase-2. In almost 50%
of IMR90-E1A caspase-9 DN cells overexpressing caspase-2, a diffuse
cytosolic staining for cytochrome c can be observed.
Caspase-2 Overexpression Triggers the Translocation of Bid to
Mitochondria--
Bid is a proapoptotic member of the Bcl-2 family of
proteins that contains only a BH3 domain (33). Bid is a substrate of caspase-8 that, once processed, translocates to mitochondria and potently induces cytochrome c release (34, 35). We therefore investigated if Bid was translocated to mitochondria in
IMR90-E1A-caspase-9 DN cells, overexpressing caspase-2.
Bid is specifically cleaved at Asp59 by caspase-8 in the
Fas apoptotic signaling pathway (34, 35). In vitro,
caspase-2 and caspase-3 can partially cleave Bid at the same aspartic
residue (34). As shown in Fig.
8a, we confirmed that both
caspase-2 and caspase-3 could cleave Bid. However, it is important to
note that the same amount of recombinant caspase-2 and caspase-3 that was able to cleave Bid only partially was sufficient to efficiently process caspase-2 (Fig. 8b).
Recently, a Bid bearing a C-terminal GFP tag has been used to monitor
its subcellular localization during apoptosis. Bid processing and its
subsequent myristoylation at glycine 60 promote its targeting to
mitochondria (36).
We have generated a similar GFP-fused Bid to analyze its subcellular
localization in IMR90-E1A-caspase-9 DN cells overexpressing caspase-2.
Caspase-8 and PLAP were also overexpressed as a control. A confocal
microscopy analysis is shown in Fig. 8c. GFP showed a
spotted localization, which was coincident with mitochondria, as
evidenced by staining with an antibody against cytochrome c oxidase (COX), in cells co-expressing caspase-2 and Bid-GFP.
On the contrary, GFP showed a diffuse nuclear/cytosolic distribution in
cells co-expressing Bid-GFP and PLAP. As expected, Bid-GFP was observed
co-localizing with mitochondria in caspase-8-overexpressing cells. Fig.
8d shows a quantitative analysis of Bid-GFP localization in
IMR90-E1A caspase-9 DN cells overexpressing caspase-2, PLAP, and
caspase-8. In almost 50% of IMR90-E1A caspase-9 DN cells
overexpressing caspase-2, Bid-GFP was translocated to mitochondria.
In this report, we have analyzed the dependence of caspase-2
processing on caspase-9 and caspase-3 during apoptosis in
vivo.
We have used human fibroblasts overexpressing catalytically inactive
caspase-9 (20) and MCF-7 cells in which wild type caspase-3 or its
catalytic inactive mutant has been reintroduced (22). Apoptosis was
induced by stimuli that activate different apoptotic pathways, namely
UV irradiation, which provokes DNA damage and mitochondrial dysfunction
(30, 31), and activation of the death receptor for TNF- By FACS analysis, we noticed that both cells lines were resistant to
UV-dependent apoptosis, in agreement with the previous results obtained using MEF from mice knock-out for caspase-9 or ES
cells To observe caspase-2 activation during apoptosis, we have produced a
rabbit antiserum that recognizes the p48-unprocessed form and the
p33-cleaved form (13). In the current study, we have followed the
appearance of the p33 and the disappearance of the p48 form as signs of
caspase-2 processing and activation.
Our data suggest that during apoptosis induced by UV irradiation,
efficient processing of caspase-2 is dependent on caspase-9 and
caspase-3.
Different results indicate that in vitro caspase-3 can
cleave caspase-2 (13, 39, 40, 41). Caspase-2 processing can be
triggered by caspase-2 itself and by caspase-3 in vitro, and it is dependent from Asp residues at position 316 (13). Caspase-2 and
caspase-3 share a strong requirement for Asp in P4 and are generally
classified in the group II caspases (42). However, some distinctions in
terms of recognition of cleaved target sequences are suggested by the
requirement of caspase-2 for a P5 residue (43).
Caspase-3-dependent processing of caspase-2 during
apoptosis in vivo has been suggested by using the inhibitor
peptide N-acetyl-Asp-Glu-Val-Asp-aldehyde (13). The
use of this inhibitor cannot exclude an effect on different caspases
such as caspase-2 itself or caspase-7. Our results, obtained by using
cell lines containing defined mutations in caspase-9 and caspase-3,
unambiguously demonstrate a critical role of caspase-3 in the
processing of caspase-2 in vivo in human cells.
The in vitro proteolytic assay suggests that among the
different caspases tested, caspase-2 was the best substrate for
caspase-3, thus reinforcing the idea that caspase-3 is the caspase
involved in caspase-2 processing during UV. In this respect, a more
detailed analysis will be necessary to specifically determine the
affinity of caspase-3 for processing the different caspases. Caspase-2 processing in response to TNF- Therefore, caspase-9 seems dispensable for caspase-2 processing in
response to TNF--dependent apoptosis, capase-2 processing is
independent of caspase-9 but still requires caspase-3. In
vitro procaspase-2 is the preferred caspase cleaved by caspase-3,
while caspase-7 cleaves procaspase-2 with reduced efficiency. We
have also demonstrated that caspase-2-mediated apoptosis requires
caspase-9 and that cells co-expressing caspase-2 and a dominant
negative form of caspase-9 are impaired in activating a normal
apoptotic response and release cytochrome c into the cytoplasm. Our findings suggest a role played by caspase-2 as a
regulator of the mitochondrial integrity and open questions on the
mechanisms responsible for its activation during cell death.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-amyloid-induced neuron death
and salmonella-induced macrophage death (9, 10).
. Recruitment of caspase-2 to the TNF-
receptor
is regulated by the interaction between the CARD domain present in the
prodomain of caspase-2 and a similar CARD domain present in RAIDD/CRADD
(11, 12).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside, bacteria where
collected by centrifugation at 3500 rpm for 5 min and lysed in 50 mM NaH2PO4, pH 8, 300 mM NaCl, 10 mM imidazole, 1 mg/ml lysozyme, 0.5 mM phenylmethylsulfonyl fluoride. After sonication, the
insoluble fraction was resuspended in sample buffer (2% SDS, 10%
glycerol, 120 mM Tris-HCl, pH 6.8, 0.005% bromphenol blue,
1%
-mercaptoethanol) and then run on an SDS-10% polyacrylamide gel. The gel was stained with Coomassie Blue R-250 to identify the
caspase-2 band, which was then electroeluted. The protein was dialyzed
overnight in PBS and purified by nickel chromatography using a His-trap
column (Qiagen), eluted with an imidazole buffer (50 mM
NaH2PO4, pH 8, 300 mM NaCl, 250 mM imidazole), and then used to immunize rabbits.
or 10 ng/ml TNF-
(MCF-7pBC3 and MCF-7pBC3mut) or with 1 µg/ml cycloheximide (Sigma) alone as a control.
argon laser and a 543
helium neon laser.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Characterization of anti-caspase-2
antibody. a, equal amounts of 293 total cell lysates
transfected with pcDNA3HAGas-2 or pCDNA3HA caspase-2 C303G were
subjected to 15% SDS-polyacrylamide gel electrophoresis, and
immunoblotting was performed using anti-caspase-2, anti-HA, or
anti-actin antibodies. b, 293 cells were UV-irradiated as
indicated. After 24 h, lysates from both adherent viable cells and
nonadherent apoptotic cells were combined and were Western blotted with
anti-caspase-2 antibody. c, different cell lines were
UV-irradiated (120 J/m2) or untreated. 20 h later,
untreated ( ) and apoptotic cells (+) were harvested. Western analysis
was performed using anti-caspase-2 antibody.
-catenin processing (19). This band
is similar in size to the previously identified intermediate processed
form of caspase-2, p33. The small and large subunits of caspase-2 were
detected only after long exposure of the blot (Fig. 1b). In
conclusion, this evidence indicates that the produced antibody
specifically recognizes caspase-2 both in normal and apoptotic cells.
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Fig. 2.
Cell cycle analysis after UV treatment.
Flow cytometric analysis of apoptosis after propidium iodide staining.
IMR90-E1A, IMR90-E1A caspase-9 DN, MCF-7 caspase-3, and MCF-7 caspase-3
CI were UV-irradiated (180-200 J/m2) and 15-18 h later
processed for the analysis.
.
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Fig. 3.
Caspase-2 processing in human fibroblasts
expressing a catalytic inactive form of caspase-9. a,
IMR90-E1A and IMR90-E1A caspase-9 DN were UV-irradiated or untreated.
After 15 h, lysates from both adherent viable cells (V)
and nonadherent dead cells (D) were harvested separately.
Western analysis was performed using anti-caspase-2, anti-PARP, or
anti-actin antibody as described. b, IMR90-E1A and IMR90-E1A
caspase-9 DN were treated with TNF- (20 ng/ml) and cycloheximide (1 µg/ml), treated with cycloheximide (1 µg/ml) alone, or left
untreated. 36 h later, adherent and nonadherent treated cells were
collected separately, while in untreated cells nonadherent apoptotic
cells were almost undetectable. Western analysis was performed using
anti-caspase-2, anti-PARP, or anti-actin antibody as a loading
control.
in the presence of
cycloheximide, and Western blot analysis was performed using lysates
prepared from TNF-
/CHX- and CHX-treated or untreated cells.
Nonadherent dead cells (Fig. 3, D) and adherent viable cells
(V) were harvested separately.
-triggered cell death, despite of the presence of a dominant
negative form of caspase-9, caspase-2 was normally processed, thus
generating the p33 fragment (Fig. 3b). PARP processing
during apoptosis induced by TNF-
was independent from caspase-9
activity. Here again, detection of actin was used as a loading control.
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Fig. 4.
Caspase-2 processing during apoptosis in
MCF-7 cells. a, MCF-7 caspase-3 and MCF-7 caspase-3 CI
were UV-irradiated or untreated. 18 h later, lysates from both
adherent viable cells and nonadherent apoptotic cells were combined.
Western analysis was performed using anti-caspase-2, anti-PARP, or
anti-actin antibody as a loading control. b, MCF-7 caspase-3
and mutated MCF-7 caspase-3 were treated with TNF- (10 ng/ml) and
cycloheximide (1 µg/ml), treated with cycloheximide (1 µg/ml)
alone, or left untreated. 36 h later, adherent and nonadherent
treated cells were collected separately, while in untreated cells
nonadherent apoptotic cells were almost undetectable. Western analysis
was performed using anti-caspase-2, anti-PARP, or anti-actin antibody
as a loading control.
-induced apoptosis. MCF-7 wild type caspase-3 and
caspase-3 CI cells were treated with TNF-
in the presence of
cycloheximide for 36 h, and Western blot analysis was performed. Nonadherent dead (Fig. 4, D) and adherent viable
(V) cells were harvested separately.
and CHX. In apoptotic MCF-7
caspase-3 CI cells, caspase-2 processing was largely impaired.
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Fig. 5.
Caspase-2 is the preferred caspase cleaved by
caspase-3 in vitro.
[35S]Methionine-labeled in vitro translated
products of the indicated caspases and PARP were incubated for 1 h
at 37 °C with increasing amounts of recombinant caspase-3 or with
caspase-3 buffer alone.
in MCF-7 cells defective for caspase-3
activity. Since caspase-3 and caspase-7 show overlapping cleavage
consensus sequences and share many death substrates in vivo
(4), we asked whether caspase-7 was able to cleave caspase-2 in an
in vitro assay.
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Fig. 6.
Processing of caspase-2 can be mediated by
caspase-7. a, [35S]methionine-labeled
in vitro translated caspase-2 and PARP were incubated for
1 h at 37 °C with increasing amounts of recombinant caspase-7
or with caspase-7 buffer alone. b, MCF-7 caspase-3 and MCF-7
caspase-3 CI were treated with TNF- (10 ng/ml) and cycloheximide (1 µg/ml), treated with cycloheximide (1 µg/ml) alone, or left
untreated. 36 h later, adherent and nonadherent treated cells were
collected separately, while in untreated cells nonadherent apoptotic
cells were almost undetectable. Western analysis was performed using
anti-caspase-7 antibodies.
/CHX.
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Fig. 7.
Apoptosis induced by caspase-2 triggers
cytochrome c release and requires caspase-9
activity. a, in IMR90-E1A caspase-9 DN and IMR90-E1A cells,
pGDSV7S-caspase-2, pGDSV7S-bax, or pGDSV7S-hPLAP was co-transfected
with pEGFPN1 as a reporter. The appearance of apoptotic cells
was scored after 20 h from transfection. b, IMR90-E1A
caspase-9 DN cells were co-transfected with
pGDSV7S-caspase-2 and pEGFPN1 as a reporter. After 20 h, an immunofluorescence assay was performed using anti-cytochrome
c antibody. c, IMR90-E1A caspase-9 DN and
IMR90-E1A cells co-expressing caspase-2 and GFP were scored for
cytochrome c release from mitochondria.
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Fig. 8.
Caspase-2 overexpression triggers the
translocation of Bid to mitochondria. a,
[35S]methionine-labeled in vitro translated
Bid was incubated for 1 h at 37 °C with recombinant caspase-2
or caspase-3. N-Acetyl-Asp-Glu-Val-Asp-aldehyde was
used at a concentration of 1 µM. b,
[35S]methionine-labeled in vitro translated
caspase-2 was incubated for 1 h at 37 °C with recombinant
caspase-2, caspase-3, or buffer alone. c, IMR90-E1A
caspase-9 DN cells were co-transfected with pGDSV7S-caspase-2 and
pEGFPN1-Bid, with pGDSV7S-PLAP and pEGFP-Bid, or with pGDSV7S-caspase-8
and pEGFPN1-Bid. After 20 h, immunofluorescence assays were
performed using anti-cytochrome c oxidase (COX)
antibody. d, IMR90-E1A caspase-9 DN cells co-expressing
caspase-2 and Bid-GFP, PLAP and Bid-GFP, or caspase-8 and Bid-GFP were
scored for translocation of Bid-GFP to mitochondria.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(37).
/
for caspase-3 (6, 38). However, some debris floating in the
medium were observed when cells containing catalytic inactive caspase-9
or caspase-3 were challenged with UV. These cell fragments probably
represent the result of a "frustrated apoptosis" that is activated
in these cell lines following extensive DNA damage and mitochondrial
dysfunction. The ability to enter some sort of frustrated apoptosis was
also confirmed by the detection of PARP. Aberrant apoptosis and PARP
cleavage has been reported also in the case of MEF from caspase-3
/
(38), thus confirming our evidence. In our cellular systems, it is
possible that UV and TNF-
trigger PARP processing through the
activation of caspase-7 (25).
was normal in the presence of mutated
caspase-9, whereas in cells expressing mutated caspase-3 cleavage it
was extremely reduced.
; on the contrary, caspase-3 is required. A
different apoptotic pathway not involving the cytochrome c
and the "apoptosome," but depending on caspase-8, might be
evoked as schematized in Fig. 9.
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Fig. 9.
A model for caspase-2 processing during UV-
or TNF- -induced apoptosis.
In vitro and in vivo studies suggest that the
limited processing of caspase-2 still detectable in TNF--treated
MCF-7 caspase-3 CI could be dependent on caspase-7. Caspase-7 can
process at some extent caspase-2 in vitro, and it is
activated, although at reduced levels, in TNF-
-treated MCF-7 cells
catalytically inactive for caspase-3.
Caspase-2 has been shown to associate through its prodomain with
RAIDD/CRADD (death adaptor molecule), involved in recruiting caspase-2
to the TNF receptor-1 signaling complex (11, 12). In principle,
RAIDD/CRADD should be responsible for inducing caspase-2 dimerization
and activation following TNF- treatment.
In our cells deprived of functional caspase-3, we were unable to
observe efficient processing of caspase-2 when apoptosis was induced by
TNF- treatment, but processing was observed in the same cellular
background following reintroduction of a functional caspase-3. There
are different explanations for this contradictory result. It is
possible that caspase-2 in MCF-7 cells is not involved in TNF-
apoptosis; alternatively, caspase-2 processing is not required for
switching on its proteolytic activity following TNF receptor-1
activation, as demonstrated for caspase-9 (44).
In this respect, it will be important to understand if unprocessed caspase-2 in complex or not with CRADD can show some proteolytic activity. In this context, it is important to note that a link has been reported between caspase-2 processing and its apoptotic activity in different experimental models (45).
As summarized in Fig. 9, if caspase-2 processing is dependent on caspase-3 it is possible that caspase-2 acts as an effector caspase and that its activation is a late event during apoptosis. The limited number of caspase-2-specific substrates so far identified counters a role of this enzyme as an effector caspase. Indeed, apart from caspase-2 itself, only recently golgin-160 has been reported to be cleaved at a specific site by caspase-2. Golgin-160 can be cleaved also, at different sites by caspase-3 and caspase-7 (16). Our limited knowledge about caspase-2 substrates makes it difficult to hypothesize a role for this caspase as an effector caspase.
Alternatively, the caspase-3-dependent processing of caspase-2 could be the result of an amplification loop, which is a common event during the apoptotic response (37, 46).
The requirement of caspase-9 during apoptosis induced by caspase-2 overexpression reinforces a role of caspase-2 as a regulative caspase. We are conscious that, although caspase-2 expression can be modulated in vivo (47), its overexpression is an artificial way to induce apoptosis. However, it represents the only possible way to selectively activate a caspase-2-specific apoptotic pathway.
We observed that in cells containing catalytic inactive caspase-9 and overexpressing caspase-2, cytochrome c was released from the mitochondria and accumulated in the cytosol. A similar behavior was observed when apoptosis was induced by Bax overexpression. This result suggests that caspase-2 can act as a regulative caspase upstream of caspase-9 by regulating mitochondrial integrity, thus triggering the release of mitochondrial components including cytochrome c, which regulate caspase-9 activation (32). A role of caspase-2 in regulating mitochondrial integrity is also supported by the ability of Bcl-2 to inhibit effectively caspase-2-mediated cell death (27).
Caspase-2 has been localized in different subcellular compartments as well as in mitochondria (16, 24, 48). However, we think that additional studies are required to establish if caspase-2 is responsible for the release of cytochrome c when localized in the mitochondria.
Bid is a proapoptotic member of the Bcl-2 family proteins that contains only a BH3 domain and can be cleaved by caspase-8 following Fas/TNF receptor-1 activation (34, 35). Caspase-8-dependent processing of Bid is required for its translocation to mitochondria, where it induces cytochrome c release. Cleaved Bid undergoes N-myristoylation on the exposed glycine 60, and this post-translation modification is required for its targeting to mitochondria (36).
We have observed that overexpression of caspase-2 can induce the translocation of Bid from cytosol to mitochondria. Therefore, it is possible that in cells overexpressing caspase-2, Bid is cleaved and myristoylated and can mediate the release of cytochrome c. In vitro caspase-2 can process, albeit weakly, Bid, thus indicating that in vivo caspase-2 might directly mediate Bid translocation to mitochondria. However, we cannot exclude the possibility that Bid translocation in response to caspase-2 overexpression is not directly regulated by caspase-2.
In conclusion, our findings focus attention on caspase-2 as a regulator
of the mitochondrial integrity and raise questions concerning the
mechanisms responsible for its activation during cell death.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Y. Lazebnik, L. Faleiro, and J. Rodriguez for IMR90-E1A, IMR90-E1A caspase-9 DN, MCF-7 wild type caspase-3, and MCF-7 caspase-3 catalytic inactive cell lines and anti-caspase-7 antiserum. We also thank F. Demarchi and C. Kuhne for carefully reading the manuscript and for helpful suggestions, A. Risso for helping with the FACS analysis, and M. Stebel for support in the generation of the anti-caspase-2 antiserum.
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FOOTNOTES |
---|
* This work was supported by a grant from the Ministero della Sanità (to C. B.).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 from the Fondazione Italiana per la Ricerca sul Cancro.
To whom correspondence should be addressed. Tel.: 0432-494382;
Fax: 0432-494301; E-mail: cbrancolini@makek.dstb.uniud.it
Published, JBC Papers in Press, March 16, 2001, DOI 10.1074/jbc.M011565200
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ABBREVIATIONS |
---|
The abbreviations used are: PBS, phosphate-buffered saline; CHX, cycloheximide; CI, catalytically inactive; DN, dominant negative; PARP, poly(ADP-ribose) polymerase; PLAP, placental alkaline phosphatase; TNF, tumor necrosis factor; FACS, fluorescence-activated cell sorting.
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